Ecdysone receptor-based inducible gene expression system

ABSTRACT

This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression More specifically, this invention relates to a novel inducible gene expression system and methods of modulating gene expression in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.

[0001] This application claims priority to co-pending U.S. provisionalapplication Serial No. 60/191,355, filed Mar. 22, 2000 and to co-pendingU.S. provisional application Serial No. 60/269,799, filed Feb. 20, 2001.

FIELD OF THE INVENTION

[0002] This invention relates to the field of biotechnology or geneticengineering. Specifically, this invention relates to the field of geneexpression. More specifically, this invention relates to a novelecdysone receptor-based inducible gene expression system and methods ofmodulating the expression of a gene within a host cell using thisinducible gene expression system.

BACKGROUND OF THE INVENTION

[0003] In the field of genetic engineering, precise control of geneexpression is a valuable tool for studying, manipulating, andcontrolling development and other physiological processes. Geneexpression is a complex biological process involving a number ofspecific protein-protein interactions. In order for gene expression tobe triggered, such that it produces the RNA necessary as the first stepin protein synthesis, a transcriptional activator must be brought intoproximity of a promoter that controls gene transcription. Typically, thetranscriptional activator itself is associated with a protein that hasat least one DNA binding domain that binds to DNA binding sites presentin the promoter regions of genes. Thus, for gene expression to occur, aprotein comprising a DNA binding domain and a transactivation domainlocated at an appropriate distance from the DNA binding domain must bebrought into the correct position in the promoter region of the gene.

[0004] The traditional transgenic approach utilizes a cell-type specificpromoter to drive the expression of a designed transgene. A DNAconstruct containing the transgene is first incorporated into a hostgenome. When triggered by a transcriptional activator, expression of thetransgene occurs in a given cell type.

[0005] Another means to regulate expression of foreign genes in cells isthrough inducible promoters. Examples of the use of such induciblepromoters include the PR1-a promoter, prokaryotic repressor-operatorsystems, immunosuppressive-immunophilin systems, and higher eukaryotictranscription activation systems such as steroid hormone receptorsystems and are described below.

[0006] The PR1-a promoter from tobacco is induced during the systemicacquired resistance response following pathogen attack. The use of PR1-amay be limited because it often responds to endogenous materials andexternal factors such as pathogens, UV-B radiation, and pollutants. Generegulation systems based on promoters induced by heat shock, interferonand heavy metals have been described (Wurn et al., 1986, Proc. Natl.Acad. Sci. USA 83:5414-5418; Arnheiter et al., 1990 Cell 62:51-61;Filmus et al., 1992 Nucleic Acids Research 20:27550-27560). However,these systems have limitations due to their effect on expression ofnon-target genes. These systems are also leaky.

[0007] Prokaryotic repressor-operator systems utilize bacterialrepressor proteins and the unique operator DNA sequences to which theybind. Both the tetracycline (“Tet”) and lactose (“Lac”)repressor-operator systems from the bacterium Escherichia coli have beenused in plants and animals to control gene expression. In the Tetsystem, tetracycline binds to the TetR repressor protein, resulting in aconformational change which releases the repressor protein from theoperator which as a result allows transcription to occur. In the Lacsystem, a lac operon is activated in response to the presence oflactose, or synthetic analogs such as isopropyl-b-D-thiogalactoside.Unfortunately, the use of such systems is restricted by unstablechemistry of the ligands, i.e. tetracycline and lactose, their toxicity,their natural presence, or the relatively high levels required forinduction or repression. For similar reasons, utility of such systems inanimals is limited.

[0008] Immunosuppressive molecules such as FK506, rapamycin andcyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc. Usingthis information, a general strategy has been devised to bring togetherany two proteins simply by placing FK506 on each of the two proteins orby placing FK506 on one and cyclosporine A on another one. A synthetichomodimer of FK506 (FK1012) or a compound resulted from fusion ofFK506-cyclosporine (FKCsA) can then be used to induce dimerization ofthese molecules (Spencer et al., 1993, Science 262:1019-24; Belshaw etal., 1996 Proc Natl Acad Sci USA 93:4604-7). Gal4 DNA binding domainfused to FKBP12 and VP16 activator domain fused to cyclophilin, andFKCsA compound were used to show heterodimerization and activation of areporter gene under the control of a promoter containing Gal4 bindingsites. Unfortunately, this system includes immunosuppressants that canhave unwanted side effects and therefore, limits its use for variousmammalian gene switch applications.

[0009] Higher eukaryotic transcription activation systems such assteroid hormone receptor systems have also been employed. Steroidhormone receptors are members of the nuclear receptor superfamily andare found in vertebrate and invertebrate cells. Unfortunately, use ofsteroidal compounds that activate the receptors for the regulation ofgene expression, particularly in plants and mammals, is limited due totheir involvement in many other natural biological pathways in suchorganisms. In order to overcome such difficulties, an alternative systemhas been developed using insect ecdysone receptors (EcR).

[0010] Growth, molting, and development in insects are regulated by theecdysone steroid hormone (molting hormone) and the juvenile hormones(Dhadialla, et al., 1998. Annu. Rev. Entomol. 43: 545-569). Themolecular target for ecdysone in insects consists of at least ecdysonereceptor (EcR) and ultraspiracle protein (USP). EcR is a member of thenuclear steroid receptor super family that is characterized by signatureDNA and ligand binding domains, and an activation domain (Koelle et al.1991, Cell, 67:59-77). EcR receptors are responsive to a number ofsteroidal compounds such as ponasterone A and muristerone A. Recently,non-steroidal compounds with ecdysteroid agonist activity have beendescribed, including the commercially available insecticidestebufenozide and methoxyfenozide that are marketed world wide by Rohmand Haas Company (see International Patent Application No.PCT/EP96/00686 and U.S. Pat. No. 5,530,028). Both analogs haveexceptional safety profiles to other organisms.

[0011] International Patent Application No. PCT/US97/05330 (WO 97/38117)discloses methods for modulating the expression of an exogenous gene inwhich a DNA construct comprising the exogenous gene and an ecdysoneresponse element is activated by a second DNA construct comprising anecdysone receptor that, in the presence of a ligand therefor, andoptionally in the presence of a receptor capable of acting as a silentpartner, binds to the ecdysone response element to induce geneexpression. The ecdysone receptor of choice was isolated from Drosophilamelanogaster. Typically, such systems require the presence of the silentpartner, preferably retinoid X receptor (RXR), in order to provideoptimum activation. In mammalian cells, insect ecdysone receptor (EcR)heterodimerizes with retinoid X receptor (RXR) and regulates expressionof target genes in a ligand dependent manner. International PatentApplication No. PCT/US98/14215 (WO 99/02683) discloses that the ecdysonereceptor isolated from the silk moth Bombyx mori is functional inmammalian systems without the need for an exogenous dimer partner.

[0012] U.S. Pat. No. 5,880,333 discloses a Drosophila melanogaster EcRand ultraspiracle (USP) heterodimer system used in plants in which thetransactivation domain and the DNA binding domain are positioned on twodifferent hybrid proteins. Unfortunately, this system is not effectivefor inducing reporter gene expression in animal cells (for comparison,see Example 1.2, below).

[0013] In each of these cases, the transactivation domain and the DNAbinding domain (either as native EcR as in International PatentApplication No. PCT/US98/14215 or as modified EcR as in InternationalPatent Application No. PCT/US97/05330) were incorporated into a singlemolecule and the other heterodimeric partners, either USP or RXR, wereused in their native state.

[0014] Drawbacks of the above described EcR-based gene regulationsystems include a considerable background activity in the absence ofligands and that these systems are not applicable for use in both plantsand animals (see U.S. Pat. No. 5,880,333). For most applications thatrely on modulating gene expression, these EcR-based systems areundesirable. Therefore, a need exists in the art for improved systems toprecisely modulate the expression of exogenous genes in both plants andanimals. Such improved systems would be useful for applications such asgene therapy, large scale production of proteins and antibodies,cell-based high throughput screening assays, functional genomics andregulation of traits in transgenic animals. Improved systems that aresimple, compact, and dependent on ligands that are relativelyinexpensive, readily available, and of low toxicity to the host wouldprove useful for regulating biological systems.

[0015] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties. However, the citationof any reference herein should not be construed as an admission thatsuch reference is available as “Prior Art” to the instant application.

SUMMARY OF THE INVENTION

[0016] The present invention relates to a novel ecdysone receptor-basedinducible gene expression system, novel receptor polynucleotides andpolypeptides for use in the novel inducible gene expression system, andmethods of modulating the expression of a gene within a host cell usingthis inducible gene expression system. In particular, Applicants'invention relates to an improved gene expression modulation systemcomprising a polynucleotide encoding a receptor polypeptide comprising atruncation mutation.

[0017] Specifically, the present invention relates to a gene expressionmodulation system comprising: a) a first gene expression cassette thatis capable of being expressed in a host cell comprising a polynucleotidethat encodes a first polypeptide comprising: i) a DNA-binding domainthat recognizes a response element associated with a gene whoseexpression is to be modulated; and ii) a ligand binding domaincomprising a ligand binding domain from a nuclear receptor; and b) asecond gene expression cassette that is capable of being expressed inthe host cell comprising a polynucleotide sequence that encodes a secondpolypeptide comprising: i) a transactivation domain; and ii) a ligandbinding domain comprising a ligand binding domain from a nuclearreceptor other than an ultraspiracle receptor; wherein the DNA bindingdomain and the transactivation domain are from a polypeptide other thanan ecdysone receptor, a retinoid X receptor, or an ultraspiraclereceptor, wherein the ligand binding domains from the first polypeptideand the second polypeptide are different and dimerize.

[0018] In a specific embodiment, the ligand binding domain of the firstpolypeptide comprises an ecdysone receptor (EcR) ligand binding domainIn another specific embodiment, the ligand binding domain of the secondpolypeptide comprises a retinoid X receptor (RXR) ligand binding domain.

[0019] In a preferred embodiment, the ligand binding domain of the firstpolypeptide comprises an ecdysone receptor ligand binding domain and theligand binding domain of the second polypeptide comprises a retinoid Xreceptor ligand binding domain.

[0020] The present invention also relates to a gene expressionmodulation system according to the invention further comprising c) athird gene expression cassette comprising: i) a response element towhich the DNA-binding domain of the first polypeptide binds; ii) apromoter that is activated by the transactivation domain of the secondpolypeptide; and iii) the gene whose expression is to be modulated.

[0021] The present invention also relates to an isolated polynucleotideencoding a truncated EcR or a truncated RXR polypeptide, wherein thetruncation mutation affects ligand binding activity or ligandsensitivity.

[0022] In particular, the present invention relates to an isolatedpolynucleotide encoding a truncated EcR or a truncated RXR polypeptidecomprising a truncation mutation that reduces ligand binding activity orligand sensitivity of said EcR or RXR polypeptide. In a specificembodiment, the present invention relates to an isolated polynucleotideencoding a truncated EcR or a truncated RXR polypeptide comprising atruncation mutation that reduces steroid binding activity or steroidsensitivity of said EcR or RXR polypeptide. In another specificembodiment, the present invention relates to an isolated polynucleotideencoding a truncated EcR or a truncated RXR polypeptide comprising atruncation mutation that reduces non-steroid binding activity ornon-steroid sensitivity of said EcR or RXR polypeptide.

[0023] The present invention also relates to an isolated polynucleotideencoding a truncated EcR or a truncated RXR polypeptide comprising atruncation mutation that enhances ligand binding activity or ligandsensitivity of said EcR or RXR polypeptide. In a specific embodiment,the present invention relates to an isolated polynucleotide encoding atruncated EcR or a truncated RXR polypeptide comprising a truncationmutation that enhances steroid binding activity or steroid sensitivityof said EcR or RXR polypeptide. In another specific embodiment, thepresent invention relates to an isolated polynucleotide encoding atruncated EcR or a truncated RXR polypeptide comprising a truncationmutation that enhances non-steroid binding activity or non-steroidsensitivity of said EcR or RXR polypeptide.

[0024] The present invention also relates to an isolated polynucleotideencoding a truncated RXR polypeptide comprising a truncation mutationthat increases ligand sensitivity of a heterodimer comprising thetruncated retinoid X receptor polypeptide and a dimerization partner. Ina specific embodiment, the dimerization partner is an ecdysone receptorpolypeptide.

[0025] The present invention also relates to an isolated polypeptideencoded by a polynucleotide according to Applicants' invention. Inparticular, the present invention relates to an isolated truncated EcRor truncated RXR polypeptide comprising a truncation mutation, whereinthe EcR or RXR polypeptide is encoded by a polynucleotide according tothe invention.

[0026] Thus, the present invention also relates to an isolated truncatedEcR or truncated RXR polypeptide comprising a truncation mutation thataffects ligand binding activity or ligand sensitivity of said EcR or RXRpolypeptide.

[0027] Applicants' invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation systemaccording to the invention. Specifically, Applicants' invention providesa method of modulating the expression of a gene in a host cellcomprising the gene to be modulated comprising the steps of: a)introducing into the host cell a gene expression modulation systemaccording to the invention; and b) introducing into the host cell aligand that independently combines with the ligand binding domains ofthe first polypeptide and the second polypeptide of the gene expressionmodulation system; wherein the gene to be expressed is a component of achimeric gene comprising: i) a response element comprising a domain towhich the DNA binding domain from the first polypeptide binds; ii) apromoter that is activated by the transactivation domain of the secondpolypeptide; and iii) the gene whose expression is to be modulated,whereby a complex is formed comprising the ligand, the firstpolypeptide, and the second polypeptide, and whereby the complexmodulates expression of the gene in the host cell.

[0028] Applicants' invention also provides an isolated host cellcomprising an inducible gene expression system according to theinvention. The present invention also relates to an isolated host cellcomprising a polynucleotide or polypeptide according to the inventionAccordingly, Applicants' invention also relates to a non-human organismcomprising a host cell according to the invention

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a Gal4 DBD-CfEcRDEFchimeric polypeptide and a second gene expression cassette encoding aVP16AD-MmRXRDEF chimeric polypeptide; prepared as described in Example 1(switch 1.1).

[0030]FIG. 2: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a Gal4 DBD-CfEcRDEFchimeric polypeptide and a second gene expression cassette encoding aVP16AD-CfUSPDEF chimeric polypeptide; prepared as described in Example 1(switch 1.2).

[0031]FIG. 3: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a Gal4 DBD-MmRXRDEFchimeric polypeptide and a second gene expression cassette encoding aVP16AD-CfEcRCDEF chimeric polypeptide; prepared as described in Example1 (switch 1.3).

[0032]FIG. 4: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a Gal4 DBD-MmRXRDEFchimeric polypeptide and a second gene expression cassette encoding aVP16AD-DmEcRCDEF chimeric polypeptide; prepared as described in Example1 (switch 1.4).

[0033]FIG. 5: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a Gal4 DBD-CfUSPDEFchimeric polypeptide and a second gene expression cassette encoding aVP16AD-CfEcRCDEF chimeric polypeptide; prepared as described in Example1 (switch 1.5).

[0034]FIG. 6: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a Gal4DBD-CfEcRDEF-VP16AD chimeric polypeptide; prepared as described inExample 1 (switch 1.6).

[0035]FIG. 7: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a VP16AD-CfEcRCDEFchimeric polypeptide; prepared as described in Example 1 (switch 1.7).

[0036]FIG. 8: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a VP16AD-DmEcRCDEFchimeric polypeptide and a second gene expression cassette encoding aMmRXR polypeptide; prepared as described in Example 1 (switch 1.8).

[0037]FIG. 9: An ecdysone receptor-based gene expression systemcomprising a first gene expression cassette encoding a VP16AD-CfEcRCDEFchimeric polypeptide and a second gene expression cassette encoding a XRpolypeptide; prepared as described in Example 1 (switch 1.9).

[0038]FIG. 10: An ecdysone receptor-based gene expression systemcomprising a gene expression cassette encoding a Gal4 DBD-CfEcRCDEFchimeric polypeptide; prepared as described in Example 1 (switch 1.10).

[0039]FIG. 11: Expression data of GAIACfEcRA/BCDEF, GAL4CfEcRCDEF,GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE truncationmutants transfected into NIH3T3 cells along with VP16MmRXRDE, pFRLUc andpTKRL plasmid DNAs.

[0040]FIG. 12: Expression data of GAIACfEcRA/BCDEF, GAL4CfEcRCDEF,GALACfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAIACfEcRDE truncationmutants transfected into 3T3 cells along with VP16MmRXRE, pFRLUc andpTKRL plasmid DNAs.

[0041]FIG. 13: Expression data of VP16MmRXRA/BCDEF, VP16mMRXRCDEF,VP16mRXRDEF, VP16 MmRXREF, VP16MmRXRBam-EF, VP16MmRXRAF2del constructstransfected into NIH3T3 cells along with GAL4CfEcRCDEF, pFRLUc and pTKRLplasmid DNAs.

[0042]FIG. 14: Expression data of VP16MmRXRCDEF, VP16MmRXRCDEF,VP16MmRXRDEF, VP16MmRXREF, VP16MmRXRBam-EF, VP16AF2del constructstransfected into NIH3T3 cells along with GAL4CfEcRDEF, pFRLUc and pTKRLplasmid DNAs.

[0043]FIG. 15: Expression data of various truncated CfEcR and MmRXRreceptor pairs transfected into NIH3T3 cells along with GAL4CfEcRDEF,pFRLUc and pTKRL plasmid DNAs.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Applicants have now developed an improved ecdysone receptor-basedinducible gene expression system comprising a truncation mutant of anecdysone receptor or a retinoid X receptor (RXR) polypeptide thataffects ligand binding activity or ligand sensitivity. This mutationaleffect may increase or reduce ligand binding activity or ligandsensitivity and may be steroid or non-steroid specific. Thus,Applicants' invention provides an improved ecdysone receptor-basedinducible gene expression system useful for modulating expression of agene of interest in a host cell. In a particularly desirable embodiment,Applicants' invention provides an inducible gene expression system thathas a reduced level of background gene expression and responds tosubmicromolar concentrations of non-steroidal ligand. Thus, Applicants'novel inducible gene expression system and its use in methods ofmodulating gene expression in a host cell overcome the limitations ofcurrently available inducible expression systems and provide the skilledartisan with an effective means to control gene expression.

[0045] The present invention provides a novel inducible gene expressionsystem that can be used to modulate gene expression in both prokaryoticand eukaryotic host cells. Applicants' invention is useful forapplications such as gene therapy, large scale production of proteinsand antibodies, cell-based high throughput screening assays, functionalgenomics and regulation of traits in transgenic organisms.

[0046] Definitions

[0047] In this disclosure, a number of terms and abbreviations are used.The following definitions are provided and should be helpful inunderstanding the scope and practice of the present invention.

[0048] In a specific embodiment, the term “about” or “approximately”means within 20%, preferably within 10%, more preferably within 5%, andeven more preferably within 1% of a given value or range.

[0049] The term “substantially free” means that a composition comprising“A” (where “A” is a single protein, DNA molecule, vector, reconbinanthost cell, etc.) is substantially free of “B” (where “B” comprises oneor more contaminating proteins, DNA molecules, vectors, etc.) when atleast about 75% by weight of the proteins, DNA, vectors (depending onthe category of species to which A and B belong) in the composition is“A”. Preferably, “A” comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

[0050] The term “isolated” for the purposes of the present inventiondesignates a biological material (nucleic acid or protein) that has beenremoved from its original environment (the environment in which it isnaturally present).

[0051] For example, a polynucleotide present in the natural state in aplant or an animal is not isolated. The same polynucleotide separatedfrom the adjacent nucleic acids in which it is naturally present. Theterm “purified” does not require the material to be present in a formexhibiting absolute purity, exclusive of the presence of othercompounds. It is rather a relative definition.

[0052] A polynucleotide is in the “purified” state after purification ofthe starting material or of the natural material by at least one orderof magnitude, preferably 2 or 3 and preferably 4 or 5 orders ofmagnitude.

[0053] A “nucleic acid” is a polymeric compound comprised of covalentlylinked subunits called nucleotides. Nucleic acid includespolyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both ofwhich may be single-stranded or double-stranded. DNA includes but is notlimited to cDNA, genomic DNA, plasmids DNA, synthetic DNA, andsemi-synthetic DNA. DNA may be linear, circular, or supercoiled.

[0054] A “nucleic acid molecule” refers to the phosphate ester polymericform of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxynbonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranologs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

[0055] The term “fragment” will be understood to mean a nucleotidesequence of reduced length relative to the reference nucleic acid andcomprising, over the common portion, a nucleotide sequence identical tothe reference nucleic acid. Such a nucleic acid fragment according tothe invention may be, where appropriate, included in a largerpolynucleotide of which it is a constituent. Such fragments comprise, oralternatively consist of, oligonucleotides ranging in length from atleast 8, 10, 12, 15, 18, 20 to 25, 30, 40, 50, 70, 80, 100, 200, 500,1000 or 1500 consecutive nucleotides of a nucleic acid according to theinvention.

[0056] As used herein, an “isolated nucleic acid fragment” is a polymerof RNA or DNA that is single- or double-stranded, optionally containngsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

[0057] A “gene” refers to an assembly of nucleotides that encode apolypeptide, and includes cDNA and genomic DNA nucleic acids. “Gene”also refers to a nucleic acid fragment that expresses a specific proteinor polypeptide, including regulatory sequences preceding (5′ noncodingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and/or coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A chimeric gene may comprise coding sequences derived fromdifferent sources and/or regulatory sequences derived from differentsources. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism A “foreign” gene or “heterologous”gene refers to a gene not normally found in the host organism, but thatis introduced into the host organism by gene transfer. Foreign genes cancomprise native genes inserted into a non-native organism, or chimericgenes. A “transgene” is a gene that has been introduced into the genomeby a transformation procedure.

[0058] “Heterologous” DNA refers to DNA not naturally located in thecell, or in a chromosomal site of the cell. Preferably, the heterologousDNA includes a gene foreign to the cell.

[0059] The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

[0060] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 infra). Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridizationStringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC. Hybridization requires that the two nucleicacids contain complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.

[0061] The term “complementary” is used to describe the relationshipbetween nucleotide bases that are capable of hybridizing to one another.For example, with respect to DNA, adenosine is complementary to thymineand cytosine is complementary to guanine. Accordingly, the instantinvention also includes isolated nucleic acid fragments that arecomplementary to the complete sequences as disclosed or used herein aswell as those substantially similar nucleic acid sequences.

[0062] In a specific embodiment, the term “standard hybridizationconditions” refers to a T_(m) of 55° C., and utilizes conditions as setforth above. In a preferred embodiment, the T_(m) is 60° C.; in a morepreferred embodiment, the T_(m) is 65° C.

[0063] Post-hybridization washes also determine stringency conditions.One set of preferred conditions uses a series of washes starting with6×SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeatedwith 2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twicewith 0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleicacids comprise complementary sequences, although depending on thestringency of the hybridization mismatches between bases are possible.

[0064] The appropriate stringency for hybridizing nucleic acids dependson the length of the nucleic acids and the degree of complementation,variables well known in the art. The greater the degree of similarity orhomology between two nucleotide sequences, the greater the value ofT_(m) for hybrids of nucleic acids having those sequences. The relativestability (corresponding to higher T_(m) of nucleic acid hybridizationsdecreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybridsof greater than 100 nucleotides in length, equations for calculatingT_(m) have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

[0065] In one embodiment the length for a hybridizable nucleic acid isat least about 10 nucleotides. Preferable a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as lengh of theprobe.

[0066] The term “probe” refers to a single-stranded nucleic acidmolecule that can base pair with a complementary single stranded targetnucleic acid to form a double-stranded molecule.

[0067] As used herein, the tern “oligonucleotide” refers to a nucleicacid, generally of at least 18 nucleotides, that is hybridizable to agenomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNAmolecule. Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. A labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. Oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a nucleic acid, or to detect the presence of anucleic acid. An oligonucleotide can also be used to form a triple helixwith a DNA molecule. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

[0068] A “primer” is an oligonucleotide that hybridizes to a targetnucleic acid sequence to create a double stranded nucleic acid regionthat can serve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

[0069] “Polymerase chain reaction” is abbreviated PCR and means an invitro method for enzymatically amplifying specific nucleic acidsequences. PCR involves a repetitive series of temperature cycles witheach cycle comprising three stages: denaturation of the template nucleicacid to separate the strands of the target molecule, annealing a singlestranded PCR oligonucleotide primer to the template nucleic acid, andextension of the annealed primer(s) by DNA polymerase. PCR provides ameans to detect the presence of the target molecule and, underquantitative or semi-quantitative conditions, to determine the relativeamount of that target molecule within the starting pool of nucleicacids.

[0070] “Reverse transcription-polymerase chain reaction” is abbreviatedRT-PCR and means an in vitro method for enzymatically producing a targetcDNA molecule or molecules from an RNA molecule or molecules, followedby enzymatic amplification of a specific nucleic acid sequence orsequences within the target cDNA molecule or molecules as describedabove. RT-PCR also provides a means to detect the presence of the targetmolecule and, under quantitative or semi-quantitative conditions, todetermine the relative amount of that target molecule within thestarting pool of nucleic acids.

[0071] A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and evensynthetic DNA sequences. If the coding sequence is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

[0072] “Open reading frame” is abbreviated ORF and means a length ofnucleic acid sequence, either DNA, cDNA or RNA, that comprises atranslation start signal or initiation codon such as an ATG or AUG, anda termination codon and can be potentially translated into a polypeptidesequence.

[0073] The term “head-to-head” is used herein to describe theorientation of two polynucleotide sequences in relation to each other.Two polynucleotides are positioned in a head-to-head orientation whenthe 5′ end of the coding strand of one polynucleotide is adjacent to the5′ end of the coding strand of the other polynucleotide, whereby thedirection of transcription of each polynucleotide proceeds away from the5′ end of the other polynucleotide. The term “head-to-head” may beabbreviated (5′)-to-(5′) and may also be indicated by the symbols (← →)or (3′←5′5′→3′).

[0074] The term “tail-to-tail” is used herein to describe theorientation of two polynucleotide sequences in relation to each other.Two polynucleotides are positioned in a tail-to-tail orientation whenthe 3′ end of the coding strand of one polynucleotide is adjacent to the3′ end of the coding strand of the other polynucleotide, whereby thedirection of transcription of each polynucleotide proceeds toward theother polynucleotide. The term “tail-to-tail” may be abbreviated(3′)-to-(3′) and may also be indicated by the symbols (→ ←) or(5′→3′3′←5′).

[0075] The term “head-to-tail” is used herein to describe theorientation of two polynucleotide sequences in relation to each other.Two polynucleotides are positioned in a head-to-tail orientation whenthe 5′ end of the coding strand of one polynucleotide is adjacent to the3′ end of the coding strand of the other polynucleotide, whereby thedirection of transcription of each polynucleotide proceeds in the samedirection as that of the other polynucleotide. The term “head-to-tail”may be abbreviated (5′)-to-(3′) and may also be indicated by the symbols(→ →) or (5′→3′5′→3′).

[0076] The term “downstream” refers to a nucleotide sequence that islocated 3′ to reference nucleotide sequence. In particular, downstreamnucleotide sequences generally relate to sequences that follow thestarting point of transcription. For example, the translation initiationcodon of a gene is located downstream of the start site oftranscription.

[0077] The term “upstream” refers to a nucleotide sequence that islocated 5′ to reference nucleotide sequence. In particular, upstreamnucleotide sequences generally relate to sequences that are located onthe 5′ side of a coding sequence or starting point of transcription. Forexample, most promoters are located upstream of the start site oftranscription.

[0078] The terms “restriction endonuclease” and “restriction enzyme”refer to an enzyme that binds and cuts within a specific nucleotidesequence within double stranded DNA.

[0079] “Homologous recombination” refers to the insertion of a foreignDNA sequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

[0080] Several methods known in the art may be used to propagate apolynucleotide according to the invention Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

[0081] A “vector” is any means for the cloning of and/or transfer of anucleic acid into a host cell. A vector may be a replicon to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, phage, cosmid, chromosome, virus) that functions as anautonomous unit of DNA replication in vivo, i.e., capable of replicationunder its own control. The term “vector” includes both viral andnonviral means for introducing the nucleic acid into a cell in vitro, exvivo or in vivo. A large nunber of vectors known in the art may be usedto manipulate nucleic acids, incorporate response elements and promotersinto genes, etc. Possible vectors include, for example, plasmids ormodified viruses including, for example bacteriophages such as lambdaderivatives, or plasmids such as PBR322 or pUC plasmid derivatives, orthe Bluescript vector. For example, the insertion of the DNA fragmentscorresponding to response elements and promoters into a suitable vectorcan be accomplished by ligating the appropriate DNA fragments into achosen vector that has complementary cohesive termini. Alternatively,the ends of the DNA molecules may be enzymatically modified or any sitemay be produced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

[0082] Viral vectors, and particularly retroviral vectors, have beenused in a wide variety of gene delivery applications in cells, as wellas living animal subjects. Viral vectors that can be used include butare not limited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNAprotein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

[0083] The term “plasmid” refers to an extra chromosomal element oftencarrying a gene that is not part of the central metabolism of the cell,and usually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

[0084] A “cloning vector” is a “replicon”, which is a unit length of anucleic acid, preferably DNA, that replicates sequentially and whichcomprises an origin of replication, such as a plasmid, phage or cosmid,to which another nucleic acid segment may be attached so as to bringabout the replication of the attached segment. Cloning vectors may becapable of replication in one cell type and expression in another(“shuttle vector”).

[0085] Vectors may be introduced into the desired host cells by methodsknown in the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; andHartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990).

[0086] A polynucleotide according to the invention can also beintroduced in vivo by lipofection. For the past decade, there has beenincreasing use of liposomes for encapsulation and transfection ofnucleic acids in vitro. Synthetic cationic lipids designed to limit thedifficulties and dangers encountered with liposome mediated transfectioncan be used to prepare liposomes for in vivo transfection of a geneencoding a marker (Felgner et al., 1987. PNAS 84:7413; Mackey, et al.,1988. Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031; and Ulmer et al.,1993. Science 259:1745-1748). The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Felgner and Ringold,1989. Science 337:387-388). Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. The use of lipofection to introduce exogenous genesinto the specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly preferred in a tissue with cellularheterogeneity, such as pancreas, liver, kidney, and the brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting (Mackey, et al., 1988, supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

[0087] Other molecules are also useful for facilitating transfection ofa nucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

[0088] It is also possible to introduce a vector in vivo as a naked DNAplasmid (see U.S. Pat. Nos. 5,693,622,5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992. Hum. Gene Ther. 3:147-154; and Wu and Wu, 1987. J. Biol.Chem. 262:4429-4432).

[0089] The term “transfection” means the uptake of exogenous orheterologous RNA or DNA by a cell. A cell has been “transfected” byexogenous or heterologous RNA or DNA when such RNA or DNA has beenintroduced inside the cell. A cell has been “transformed” by exogenousor heterologous RNA or DNA when the transfected RNA or DNA effects aphenotypic change. The transforming RNA or DNA can be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

[0090] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

[0091] The term “genetic region” will refer to a region of a nucleicacid molecule or a nucleotide sequence that comprises a gene encoding apolypeptide.

[0092] In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

[0093] The term “selectable marker” means an identifying factor, usuallyan antibiotic or chemical resistance gene, that is able to be selectedfor based upon the marker gene's effect, i.e., resistance to anantibiotic, resistance to a herbicide, colorimetric markers, enzymes,fluorescent markers, and the like, wherein the effect is used to trackthe inheritance of a nucleic acid of interest and/or to identify a cellor organism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

[0094] The term “reporter gene” means a nucleic acid encoding anidentifying factor that is able to be identified based upon the reportergene's effect, wherein the effect is used to track the inheritance of anucleic acid of interest, to identify a cell or organism that hasinherited the nucleic acid of interest, and/or to measure geneexpression induction or transcription. Examples of reporter genes knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

[0095] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters”. Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

[0096] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

[0097] A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

[0098] “Transcriptional and translational control sequences” are DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

[0099] The term “response element” means one or more cis-acting DNAelements which confer responsiveness on a promoter mediated throughinteraction with the DNA-binding domains of the first chimeric gene.This DNA element may be either palindromic (perfect or imperfect) in itssequence or composed of sequence motifs or half sites separated by avariable number of nucleotides. The half sites can be similar oridentical and arranged as either direct or inverted repeats or as asingle half site or multimers of adjacent half sites in tandem. Theresponse element may comprise a minimal promoter isolated from differentorganisms depending upon the nature of the cell or organism into whichthe response element will be incorporated. The DNA binding domain of thefirst hybrid protein binds, in the presence or absence of a ligand, tothe DNA sequence of a response element to initiate or suppresstranscription of downstream gene(s) under the regulation of thisresponse element Examples of DNA sequences for response elements of thenatural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (see Cherbas L.,et. al., (1991), Genes Dev. 5, 120-131); AGGTCAN_((n))AGGTCA, whereN_((n)) can be one or more spacer nucleotides (see D'Avino P P., et.al., (1995), Mol. Cell. Endocrinol, 113, 1-9); and GGGTTGAATGAATTT (seeAntoniewski C., et. al., (1994). Mol. Cell Biol. 14, 4465-4474).

[0100] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

[0101] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from a nucleic acid or polynucleotide. Expression may also referto translation of mRNA into a protein or polypeptide.

[0102] The terms “cassette”, “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

[0103] For purposes of this invention, the term “gene switch” refers tothe combination of a, response element associated with a promoter, andan EcR based system which, in the presence of one or more ligands,modulates the expression of a gene into which the response element andpromoter are incorporated.

[0104] The terms “modulate” and “modulates” mean to induce, reduce orinhibit nucleic acid or gene expression, resulting in the respectiveinduction, reduction or inhibition of protein or polypeptide production.

[0105] The plasmids or vectors according to the invention may furthercomprise at least one promoter suitable for driving expression of a genein a host cell. The term “expression vector” means a vector, plasmid orvehicle designed to enable the expression of an inserted nucleic acidsequence following transformation into the host The cloned gene, i.e.,the inserted nucleic acid sequence, is usually placed under the controlof control elements such as a promoter, a minimal promoter, an enhancer,or the like. Initiation control regions or promoters, which are usefulto drive expression of a nucleic acid in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to: viral promoters, plantpromoters, bacterial promoters, animal promoters, mammalian promoters,synthetic promoters, constitutive promoters, tissue specific promoter,developmental specific promoters, inducible promoters, light regulatedpromoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1,TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful forexpression in Saccharomyces); AOX1 promoter (useful for expression inPichia); b-lactamase, lac, ara, tet, tryp, lP_(L), lP_(R), T7, tac, andtrc promoters (useful for expression in Escherichia coli); and lightregulated-, seed specific-, pollen specific-, ovary specific-,pathogenesis or disease related-, cauliflower mosaic virus 35S, CMV 35Sminimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b bindingprotein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, rootspecific, chitinase, stress inducible, rice tungro bacilliform virus,plant superpromoter, potato leucine aminopeptidase, nitrate reductase,mannopine synthase, nopaline synthase, ubiquitin, zein protein, andanthocyanin promoters (useful for expression in plant cells); animal andmammalian promoters known in the art include, but are not limited to,the SV40 early (SV40e) promoter region, the promoter contained in the 3′long termnal repeat (LTR) of Rous sarcoma virus (RSV), the promoters ofthe E1A or major late promoter (MLP) genes of adenoviruses, thecytomegalovirus early promoter, the herpes simplex virus (HSV) thymidinekinase (TK) promoter, an elongation factor 1 alpha (EF1) promoter, aphosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, analbumin promoter, the regulatory sequences of the mousemetallothionein-L promoter, and transcriptional control regions, theubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP, and the like), the promoters of therapeutic genes (of theMDR, CFTR or factor VIII type, and the like), and promoters that exhibittissue specificity and have been utilized in transgenic animals, such asthe elastase I gene control region which is active in pancreatic acinarcells; insulin gene control region active in pancreatic beta cells,immunoglobulin gene control region active in lymphoid cells, mousemammary tumor virus control region active in testicular, breast,lymphoid and mast cells; albumin gene, Apo AI and Apo AII controlregions active in liver, alpha-fetoprotein gene control region active inliver, alpha 1-antitrypsin gene control region active in the liver,beta-globin gene control region active in myeloid cells, myelin basicprotein gene control region active in oligodendrocyte cells in thebrain, myosin light chain-2 gene control region active in skeletalmuscle, and gonadotropic releasing hormone gene control region active inthe hypothalamus, pyruvate kinase promoter, villin promoter, promoter ofthe fatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In a preferred embodiment of the invention,the promoter is selected from the group consisting of a cauliflowermosaic virus ³⁵S promoter, a cassaya vein mosaic virus promoter, and acauliflower mosaic virus ³⁵S minimal promoter, an elongation factor 1alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, aubiquitin (Ubc) promoter, and an albumin promoter. In addition, theseexpression sequences may be modified by addition of enhancer orregulatory sequences and the like.

[0106] Enhancers that may be used in embodiments of the inventioninclude but are not limited to: tobacco mosaic virus enhancer,cauliflower mosaic virus ³⁵S enhancer, tobacco etch virus enhancer,ribulose 1,5-bisphosphate carboxylase enhancer, rice tungro bacilliformvirus enhancer, and other plant and viral gene enhancers, and the like.

[0107] Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In a preferred embodiment ofthe invention, the termination control region may be comprise or bederived from a synthetic sequence, synthetic polyadenylation signal, anSV40 late polyadenylation signal, an SV40 polyadenylation signal, abovine growth hormone (BGH) polyadenylation signal, nopaline synthase(nos), cauliflower mosaic virus (CaMV), octopine synthase (ocs),Agrocateum, viral, and plant terminator sequences, or the like.

[0108] The terms “3′ non-coding sequences” or “3′ untranslated region(UTR)” refer to DNA sequences located downstream (3′) of a codingsequence and may comprise polyadenylation [poly(A)] recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

[0109] “Regulatory region” means a nucleic acid sequence which regulatesthe expression of a second nucleic acid sequence. A regulatory regionmay include sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

[0110] A regulatory region from a “heterologous source” is a regulatoryregion that is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur m nature, but which are designed by one having ordinary skill inthe art.

[0111] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

[0112] A “polypeptide” is a polymeric compound comprised of covalentlylinked amino acid residues. Amino acids have the following generalstructure:

[0113] Amino acids are classified into seven groups on the basis of theside chain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. A polypeptide of the invention preferably comprises at leastabout 14 amino acids.

[0114] A “protein” is a polypeptide that performs a structural orfunctional role in a living cell.

[0115] An “isolated polypeptide” or “isolated protein” is a polypeptideor protein that is substantially free of those compounds that arenormally associated therewith in its natural state (e.g., other proteinsor polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” isnot meant to exclude artificial or synthetic mixtures with othercompounds, or the presence of impurities which do not interfere withbiological activity, and which may be present, for example, due toincomplete purification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

[0116] “Fragment” of a polypeptide according to the invention will beunderstood to mean a polypeptide whose amino acid sequence is shorterthan that of the reference polypeptide and which comprises, over theentire portion with these reference polypeptides, an identical aminoacid sequence. Such fragments may, where appropriate, be included in alarger polypeptide of which they are a part. Such fragments of apolypeptide according to the invention may have a length of 10, 15, 20,30 to 40, 50, 100, 200 or 300 amino acids.

[0117] A “variant” of a polypeptide or protein is any analogue,fragment, derivative, or mutant which is derived from a polypeptide orprotein and which retains at least one biological property of thepolypeptide or protein. Different variants of the polypeptide or proteinmay exist in nature. These variants may be allelic variationscharacterized by differences in the nucleotide sequences of thestructural gene coding for the protein, or may involve differentialsplicing or post-translational modification. The skilled artisan canproduce variants having single or multiple amino acid substitutions,deletions, additions, or replacements. These variants may include, interalia: (a) variants in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids, (b)variants in which one or more amino acids are added to the polypeptideor protein, (c) variants in which one or more of the amino acidsincludes a substituent group, and (d) variants in which the polypeptideor protein is fused with another polypeptide such as serum albumin. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art. Avariant polypeptide preferably comprises at least about 14 amino acids.

[0118] A “heterologous protein” refers to a protein not naturallyproduced in the cell.

[0119] A “mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

[0120] The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

[0121] A “signal sequence” is included at the beginning of the codingsequence of a protein to be expressed on the surface of a cell. Thissequence encodes a signal peptide, N-terminal to the mature polypeptide,that directs the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

[0122] The term “hornology” refers to the percent of identity betweentwo polynucleotide or two polypeptide moieties. The correspondencebetween the sequence from one moiety to another can be determined bytechniques known to the art. For example, homology can be determined bya direct comparison of the sequence information between two polypeptidemolecules by aligning the sequence information and using readilyavailable computer programs. Alternatively, homology can be determinedby hybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

[0123] As used herein, the term “homologous” in all its grammaticalforms and spelling variations refers to the relationship betweenproteins that possess a “common evolutionary origin,” including proteinsfrom superfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50:667.). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity.

[0124] Accordingly, the term “sequence similarity” in all itsgrammatical forms refers to the degree of identity or correspondencebetween nucleic acid or amino acid sequences of proteins that may or maynot share a common evolutionary origin (see Reeck et al., 1987, Cell50:667). As used herein, the term “homologous” in all its grammaticalforms and spelling variations refers to the relationship betweenproteins that possess a “common evolutionary origin,” including proteinsfrom superfamilies and homologous proteins from different species (Reecket al., supra). Such proteins (and their encoding genes) have sequencehomology, as reflected by their high degree of sequence similarity.However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and not a common evolutionary origin.

[0125] In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95To) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., 1989, supra.

[0126] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the protein encoded by the DNA sequence.“Substantially similar” also refers to nucleic acid fragments whereinchanges in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression byantisense or co-suppression technology. “Substantially similar” alsorefers to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotide basesthat do not substantially affect the functional properties of theresulting transcript. It is therefore understood that the inventionencompasses more than the specific exemplary sequences. Each of theproposed modifications is well within the routine skill in the art, asis determination of retention of biological activity of the encodedproducts.

[0127] Moreover, the skilled artisan recognizes that substantiallysimilar sequences encompassed by this invention are also defined bytheir ability to hybridize, under stringent conditions (0.1×SSC, 0.1%SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%SDS), with the sequences exemplified herein. Substantially similarnucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are at least 70% identical to the DNAsequence of the nucleic acid fragments reported herein. Preferredsubstantially nucleic acid fragments of the instant invention are thosnucleic acid fragments whose DNA sequences are at least 80% identical tothe DNA sequence of the nucleic acid fragments reported herein. Morepreferred nucleic acid fragments are at least 90% identical to the DNAsequence of the nucleic acid fragments reported herein. Even morepreferred are nucleic acid fragments that are at least 95% identical tothe DNA sequence of the nucleic acid fragments reported herein.

[0128] Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

[0129] The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

[0130] A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also wwwncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

[0131] The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0132] The term “sequence analysis software” refers to any computeralgorithm or software program that is useful for the analysis ofnucleotide or amino acid sequences. “Sequence analysis software” may becommercially available or independently developed. Typical sequenceanalysis software will include but is not limited to the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters whichoriginally load with the software when first initialized.

[0133] “Synthetic genes” can be assembled from oligonucleotide buildingblocks that are chemically synthesized using procedures known to thoseskilled in the art. These building blocks are ligated and annealed toform gene segments that are then enzymatically assembled to constructthe entire gene. “Chemically synthesized”, as related to a sequence ofDNA, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of DNA may be accomplished using wellestablished procedures, or automated chemical synthesis can be performedusing one of a number of commercially available machines. Accordingly,the genes can be tailored for optimal gene expression based onoptimization of nucleotide sequence to reflect the codonbias of the hostcell. The skilled artisan appreciates the likelihood of successful geneexpression if codon usage is biased towards those codons favored by thehost. Determination of preferred codons can be based on a survey ofgenes derived from the host cell where sequence information isavailable.

[0134] Gene Expression Modulation System of the Invention

[0135] Applicants have now shown that separating the transactivation andDNA binding domains by placing them on two different proteins results ingreatly reduced background activity in the absence of a ligand andsignificantly increased activity over background in the presence of aligand. Applicants' improved gene expression system comprises twochimeric gene expression; the first encoding a DNA binding domain fusedto a nuclear receptor polypeptide and the second encoding atransactivation domain fused to a nuclear receptor polypeptide. Theinteraction of the first protein with the second protein effectivelytethers the DNA binding domain to the transactivation domain. Since theDNA binding and transactivation domains reside on two differentmolecules, the background activity in the absence of ligand is greatlyreduced.

[0136] In general, the inducible gene expression modulation system ofthe invention comprises a) a first chimeric gene that is capable ofbeing expressed in a host cell comprising a polynucleotide sequence thatencodes a first hybrid polypeptide comprising i) a DNA-binding domainthat recognizes a response element associated with a gene whoseexpression is to be modulated; and ii) a ligand binding domaincomprising the ligand binding domain from a nuclear receptor; and b) asecond chimeric gene that is capable of being expressed in the host cellcomprising a polynucleotide sequence that encodes a second hybridpolypeptide comprising: i) a transactivation domain; and ii) a ligandbinding domain comprising the ligand binding domain from a nuclearreceptor other than ultraspiracle (USP); wherein the transactivationdomain are from other than EcR, RXR, or USP; and wherein the ligandbinding domains from the first hybrid polypeptide and the second hybridpolypeptide are different and dimerize.

[0137] This two-hybrid system exploits the ability of a pair ofinteracting proteins to bring the transcription activation domain into amore favorable position relative to the DNA binding domain such thatwhen the DNA binding domain binds to the DNA binding site on the gene,the transactivation domain more effectively activates the promoter (see,for example, U.S. Pat. No. 5,283,173). This two-hybrid system is asignificantly improved inducible gene expression modulation systemcompared to the two systems disclosed in International PatentApplications PCT/US97/05330 and PCT/US98/14215. The ecdysonereceptor-based gene expression modulation system of the invention may beeither heterodimeric and homodimeric. A functional EcR complex generallyrefers to a heterodimeric protein complex consisting of two members ofthe steroid receptor family, an ecdysone receptor protein obtained fromvarious insects, and an ultraspiracle (USP) protein or the vertebratehomolog of USP, retinoid X receptor protein (see Yao, et al. (1993)Nature 366, 476-479; Yao, et al., (1992) Cell 71, 63-72). However, thecomplex may also be a homodimer as detailed below. The functionalecdysteroid receptor complex may also include additional protein(s) suchas immunophilins. Additional members of the steroid receptor family ofproteins, known as transcriptional factors (such as DHR38 or betaFTZ-1),may also be ligand dependent or independent partners for EcR, USP,and/or RXR. Additionally, other cofactors may be required such asproteins generally known as coactivators (also termed adapters ormediators). These proteins do not bind sequence-specifically to DNA andare not involved in basal transcription. They may exert their effect ontranscription activation through various mechanisms, includingstimulation of DNA-binding of activators, by affecting chromatinstructure, or by mediating activator-initiation complex interactions.Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70,SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as thepromiscuous coactivator C response element B binding protein, CBP/p300(for review see Glass et al, Curr. Opin. Cell Biol. 9:222-232, 1997).Also, protein cofactors generally known as corepressors (also known asrepressors, silencers, or silencing mediators) may be required toeffectively inhibit transcriptional activation in the absence of ligand.These corepressors may interact with the ullliganded ecdysone receptorto silence the activity at the response element Current evidencesuggests that binding of ligand changes the conformation of thereceptor, which results in release of the corepressor and recruitment ofthe above descnbed coactivators, thereby abolishing their silencingactivity. Examples of corepressors include N-CoR and SMRT (for review,see Horwitz et al. Mol Endocrinol. 10: 1167-1177, 1996). These cofactorsmay either be endogenous within the cell or organism, or may be addedexogenously as transgenes to be expressed in either a regulated orunregulated fashion. Homodimer complexes of the ecdysone receptorprotein, USP, or RXR may also be functional under some circumstances.

[0138] The ecdysone receptor complex typically includes proteins whichare members of the nuclear receptor superfamily wherein all members arecharacterized by the presence of an amino-terminal transactivationdomain, a DNA binding domain (“DBD”), and a ligand binding domain(“LBD”) separated from the DBD by a hinge region. As used herein, theterm “DNA binding domain” comprises a minimal peptide sequence of a DNAbinding protein, up to the entire length of a DNA binding protein, solong as the DNA binding domain functions to associate with a particularresponse element. Members of the nuclear receptor superfamily are alsocharacterized by the presence of four or five domains: A/B, C, D, E, andin some members F (see Evans, Science 240:889-895 (1988)). The “A/B”domain corresponds to the transactivation domain, “C” corresponds to theDNA-binding domain, “D” corresponds to the hinge region, and “E”corresponds to the ligand binding domain. Some members of the family mayalso have another transactivation domain on the carboxy-terminal side ofthe LBD corresponding to “F”.

[0139] The DBD is characterized by the presence of two cysteine zincfingers between which are two amino acid motifs, the P-box and theD-box, which confer specificity for ecdysone response elements. Thesedomains may be either native, modified, or chimeras of different domainsof heterologous receptor proteins. This EcR receptor, like a subset ofthe steroid receptor family, also possesses less well defined regionsresponsible for heterodimerization properties. Because the domains ofEcR, USP, and RXR are modular in nature, the LBD, DBD, andtransactivation domains may be interchanged.

[0140] Gene switch systems are known that incorporate components fromthe ecdysone receptor complex. However, in these known systems, wheneverEcR is used it is associated with native or modified DNA binding domainsand transactivation domains on the same molecule. USP or RXR aretypically used as silent partners. We have now shown that when DNAbinding domains and transactivation domains are on the same molecule thebackground activity in the absence of ligand is high and that suchactivity is dramatically reduced when DNA binding domains andtransactivation domains are on different molecules, that is, on each oftwo partners of a heterodimeric or homodimeric complex. This two-hybridsystem also provides improved sensitivity to non-steroidal ligands forexample, diacylhydrazines, when compared to steroidal ligands forexample, ponasterone A (“PonA”) or muristegone A (“MurA”). That is, whencompared to steroids, the non-steroidal ligands provide higher activityat a lower concentration. In addition, since transactivation based onEcR gene switches is often cell-line dependent, it is easier to tailorswitching system to obtain maximum transactivation capability for eachapplication. Furthermore, this two-hybrid system avoids some sideeffects due to overexpression of RXR that often occur when unmodifiedRXR is used as a switching partner. In this two-hybrid system, nativeDNA binding and transactivation domains of EcR or RXR are eliminated. Asa result, these chimeric molecules have less chance of interacting withother steroid hormone receptors present in the cell resulting in reducedside effects.

[0141] Specifically, Applicants' invention relates to a gene expressionmodulation system comprising: a) a first gene expression cassette thatis capable of being expressed in a host cell, wherein the first geneexpression cassette comprises a polynucleotide that encodes a firstpolypeptide comprising i) a DNA-binding domain that recognizes aresponse element associated with a gene whose expression is to bemodulated; and ii) a ligand binding domain comprising a ligand bindingdomain from a nuclear receptor; and b) a second gene expression cassettethat is capable of being expressed in the host cell, wherein the secondgene expression cassette comprises a polynucleotide sequence thatencodes a second polypeptide comprising i) a transactivation domain; andii) a ligand binding domain comprising a ligand binding domain from anuclear receptor other than ultraspiracle (USP); wherein the DNA bindingdomain and the transactivation domain are from other than EcR, RXR, orUSP; wherein the ligand binding domains from the first polypeptide andthe second polypeptide are different and dimerize.

[0142] The present invention also relates to a gene expressionmodulation system according to the present invention further comprisingc) a third gene expression cassette comprising: i) the response elementto which the DNA-binding domain of the first polypeptide binds; ii) apromoter that is activated by the transactivation domain of the secondpolypeptide; and iii) the gene whose expression is to be modulated.

[0143] In a specific embodiment, the gene whose expression is to bemodulated is a homologous gene with respect to the host cell. In anotherspecific embodiment, the gene whose expression is to be modulated is aheterologous gene with respect to the host cell.

[0144] In a specific embodiment, the ligand binding domain of the firstpolypeptide comprises an ecdysone receptor ligand binding domain.

[0145] In another specific embodiment, the ligand binding domain of thefirst polypeptide comprises a retinoid X receptor ligand binding domain.

[0146] In a specific embodiment, the ligand binding domain of the secondpolypeptide comprises an ecdysone receptor ligand binding domain.

[0147] In another specific embodiment, the ligand binding domain of thesecond polypeptide comprises a retinoid X receptor ligand bindingdomain.

[0148] In a preferred embodiment, the ligand binding domain of the firstpolypeptide comprises an ecdysone receptor ligand binding domain, andthe ligand binding domain of the second polypeptide comprises a retinoidX receptor ligand binding domain.

[0149] In another preferred embodiment, the ligand binding domain of thefirst polypeptide is from a retinoid X receptor polypeptide, and theligand binding domain of the second polypeptide is from an ecdysonereceptor polypeptide.

[0150] Preferably, the ligand binding domain is an EcR or RXR relatedsteroid/thyroid hormone nuclear receptor family member ligand bindingdomain, or analogs, combinations, or modifications thereof. Morepreferably, the LBD is from EcR or RXR. Even more preferably, the LBD isfrom a truncated EcR or RXR. A truncation mutation may be made by anymethod used in the art, including but not limited to restrictionendonuclease digestion/deletion, PCR-mediated/oligonucleotide directeddeletion, chemical mutagenesis, UV strand breakage, and the like.

[0151] Preferably, the EcR is an insect EcR selected from the groupconsisting of a Lepidopteran EcR, a Dipteran EcR, an Arthropod EcR, aHomopteran EcR and a Hemipteran EcR. More preferably, the EcR for use isa spruce budworm Choristoneura fumiferana EcR (“CfEcR”), a Tenebriomolitor EcR (“TmEcR”), a Manduca sexta EcR (“MsEcR”), a Heliothiesvirescens EcR (“HvEcR”), a silk moth Bombyx mori EcR (“BnEcR”), a fruitfly Drosophila melanogaster EcR (“DmEcR”), a mosquito Aedes aegypti EcR(“AaEcR”), a blowfly Lucilia capitata EcR (“LcEcR”), a Mediterraneanfruit fly Ceratitis capitata EcR (“CcEcR”), a locust Locusta migratoriaEcR (“LmEcR”), an aphid Myzus persicae EcR (“MpEcR”), a fiddler crab Ucapugilator EcR (“UpEcR”), or an ixodid tick Amblyomma americanum EcR(“AmaEcR”). Even more preferably, the LBD is from spruce budworm(Choristoneura fumiferana) EcR (“CfEcR”) or fruit fly Drosophilamelanogaster EcR (“DmEcR”).

[0152] Preferably, the LBD is from a truncated insect EcR. The insectEcR polypeptide truncation comprises a deletion of at least 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, theinsect EcR polypeptide truncation comprises a deletion of at least apartial polypeptide domain. Even more preferably, the insect EcRpolypeptide truncation comprises a deletion of at least an entirepolypeptide domain. In a specific embodiment, the insect EcR polypeptidetruncation comprises a deletion of at least an A/B-domain deletion, aC-domain deletion, a D-domain deletion, an E-domain deletion, anF-domain deletion, an A/B/C-domains deletion, an A/B/1/2-C-domainsdeletion, an AIB/C/Ddomains deletion, an A/B/C/D/F-domains deletion, anA/B/F-domains, and an A/B/C/F-domains deletion. A combination of severalcomplete and/or partial domain deletions may also be performed.

[0153] In a preferred embodiment, the ecdysone receptor ligand bindingdomain is encoded by a polynucleotide comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0154] In another preferred embodiment, the ecdysone receptor ligandbinding domain comprises a polypeptide sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, and SEQ ID NO: 20. Preferably, the RXR polypeptide is a mouseMus musculus RXR (“MmRXR”) or a human Homo sapiens RXR (“HsRXR”). TheRXR polypeptide may be an RXR_(α), RXR_(β), or RXR_(γ) isoform.

[0155] Preferably, the LBD is from a truncated RXR. The RXR polypeptidetruncation comprises a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, or 265 amino acids. More preferably, the RXR polypeptidetruncation comprises a deletion of at least a partial polypeptidedomain. Even more preferably, the RXR polypeptide truncation comprises adeletion of at least an entire polypeptide domain. In a specificembodiment, the RXR polypeptide truncation comprises a deletion of atleast an A/B-domain deletion, a C-domain deletion, a D-domain deletion,an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion,an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, anA/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domainsdeletion. A combination of several complete and/or partial domaindeletions may also be performed.

[0156] In a preferred embodiment, the retinoid X receptor ligand bindingdomain is encoded by a polynucleotide comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0157] In another preferred embodiment, the retinoid X receptor ligandbinding domain comprises a polypeptide sequence selected from the groupconsisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO: 40.

[0158] For purposes of this invention EcR and RXR also include syntheticand chimeric EcR and RXR and their homologs.

[0159] The DNA binding domain can be any DNA binding domain with a knownresponse element, including synthetic and chimeric DNA binding domains,or analogs, combinations, or modifications thereof. Preferably, the DBDis a GAL4 DBD, a LexA DBD, a transcription factor DBD, a steroid/thyroidhormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD,or a yeast put DBD. More preferably, the DBD is a GAL4 DBD [SEQ ID NO:41 (polynucleotide) or SEQ ID NO: 42 (polypeptide)] or a LexA DBD [(SEQID NO: 43 (polynucleotide) or SEQ ID NO: 44 (polypeptide)].

[0160] The transactivation domain (abbreviated “AD” or “TA”) may be anysteroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD,polyglutamine AD, basic or acidic amino acid AD, a VP16 AD, a GAL4 AD,an NF-κB AD, a BP64 AD, or an analog, combination, or modificationthereof. Preferably, the AD is a synthetic or chimeric AD, or isobtained from a VP16, GAL4, or NF-kB. Most preferably, the AD is a VP16AD [SEQ ID NO: 45 (polynucleotide) or SEQ ID NO: 46 (polypeptide)].

[0161] The response element (“RE”) may be any response element with aknown DNA binding domain, or an analog, combination, or modificationthereof. Preferably, the RE is an RE from GALA (“GAL4RE”), LexA, asteroid/thyroid hormone nuclear receptor RE, or a synthetic RE thatrecognizes a synthetic DNA binding domain. More preferably, the RE is aGAL4RE comprising a polynucleotide sequence of SEQ ID NO: 47 or a LexA8× operon comprising a polynucleotide sequence of SEQ ID NO: 48.Preferably, the first hybrid protein is substantially free of atransactivation domain and the second hybrid protein is substantiallyfree of a DNA binding domain. For purposes of this invention,“substantially free” means that the protein in question does not containa sufficient sequence of the domain in question to provide activation orbinding activity.

[0162] The ligands for use in the present invention as described below,when combined with the ligand binding domain of an EcR, USP, RXR, oranother polypeptide which in turn are bound to the response elementlinked to a gene, provide the means for external temporal regulation ofexpression of the gene. The binding mechanism or the order in which thevarious components of this invention bind to each other, that is, ligandto receptor, first polypeptide to response element, second polypeptideto promoter, etc., is not critical. Binding of the ligand to the ligandbinding domains of an EcR, USP, RXR, or another protein, enablesexpression or suppression of the gene. This mechanism does not excludethe potential for ligand binding to EcR, USP, or RXR, and the resultingformation of active homodimer complexes (e.g. EcR+EcR or USP+USP).Preferably, one or more of the receptor domains can be varied producinga chimeric gene switch. Typically, one or more of the three domains,DBD, LBD, and transactivation domain, may be chosen from a sourcedifferent than the source of the other domains so that the chimericgenes and the resulting hybrid proteins are optimized in the chosen hostcell or organism for transactivating activity, complementary binding ofthe ligand, and recognition of a specific response element In addition,the response element itself can be modified or substituted with responseelements for other DNA binding protein domains such as the GAL4 proteinfrom yeast (see Sadowski, et al. (1988) Nature, 335:563-564) or LexAprotein from E. coli (see Brent and Ptashne (1985), Cell, 43:729-736),or synthetic response elements specific for targeted interactions withproteins desigped, modified, and selected for such specific interactions(see, for example, Kim, et al. (1997), Proc. Natl. Acad. Sci., USA,94:3616-3620) to accommodate chimeric receptors. Another advantage ofchimeric systems is that they allow choice of a promoter used to drivethe gene expression according to a desired end result Such doublecontrol can be particularly important in areas of gene therapy,especially when cytotoxic proteins are produced, because both the timingof expression as well as the cells wherein expression occurs can becontrolled. When genes, operatively linked to a suitable promoter, areintroduced into the cells of the subject, expression of the exogenousgenes is controlled by the presence of the system of this invention.Promoters may be constitutively or inducibly regulated or may betissue-specific (that is, expressed only in a particular type of cells)or specific to certain developmental stages of the organism.

[0163] Gene Expression Cassettes of the Invention

[0164] The novel ecdysone receptor-based inducible gene expressionsystem of the invention comprises a novel gene expression cassette thatis capable of being expressed in a host cell, wherein the geneexpression cassette comprises a polynucleotide encoding a hybridpolypeptide. Thus, Applicants' invention also provides novel geneexpression cassettes for use in the gene expression system of theinvention.

[0165] Specifically, the present invention provides a gene expressioncassette comprising a polynucleotide encoding a hybrid polypeptide. Thehybrid polypeptide comprises either 1) a DNA-binding domain thatrecognizes a response element and a ligand binding domain of a nuclearreceptor or 2) a transactivation domain and a ligand binding domain of anuclear receptor, wherein the transactivation domain is from a nuclearreceptor other than an EcR, an RXR, or a USP.

[0166] In a specific embodiment, the gene expression cassette encodes ahybrid polypeptide comprising a DNA-binding domain that recognizes aresponse element and an ecdysone receptor ligand binding domain, whereinthe DNA binding domain is from a nuclear receptor other than an ecdysonereceptor.

[0167] In another specific embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a DNA-binding domain thatrecognizes a response element and a retinoid X receptor ligand bindingdomain, wherein the DNA binding domain is from a nuclear receptor otherthan a retinoid X receptor.

[0168] The DNA binding domain can be any DNA binding domain with a knownresponse element, including synthetic and chimeric DNA binding domains,or analogs, combinations, or modifications thereof. Preferably, the DBDis a GALA DBD, a LexA DBD, a transcription factor DBD, a steroid/thyroidhormone nuclear receptor superfamily member DBD, a bacterial LacZ DBD,or a yeast put DBD. More preferably, the DBD is a GAL4 DBD [SEQ ID NO:41 (polynucleotide) or SEQ ID NO: 42 (polypeptide)] or a LexA DBD [(SEQID NO: 43 (polynucleotide) or SEQ ID NO: 44 (polypeptide)].

[0169] In another specific embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a transactivation domain and anecdysone receptor ligand binding domain, wherein the transactivationdomain is from a nuclear receptor other than an ecdysone receptor.

[0170] In another specific embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a transactivation domain and aretinoid X receptor ligand binding domain, wherein the transactivationdomain is from a nuclear receptor other than a retinoid X receptor.

[0171] The transactivation domain (abbreviated “AD” or “TA”) may be anysteroid/thyroid hormone nuclear receptor AD, synthetic or chimeric AD,polyglutamine AD, basic or acidic amino acid AD, a VPl6 AD, a GALA AD,an NF-κB AD, a BP64 AD, or an analog, combination, or modificationthereof. Preferably, the AD is a synthetic or chimeric AD, or isobtained from a VP16, GAL4, or NF-kB. Most preferably, the AD is a VP16AD [SEQ ID NO: 45 (polynucleotide) or SEQ ID NO: 46 (polypeptide)].

[0172] Preferably, the ligand binding domain is an EcR or RXR relatedsteroid/thyroid hormone nuclear receptor family member ligand bindingdomain, or analogs, combinations, or modifications thereof. Morepreferably, the LBD is from EcR or RXR. Even more preferably, the LBD isfrom a truncated EcR or RXR.

[0173] Preferably, the EcR is an insect EcR selected from the groupconsisting of a Lepidopteran EcR, a Dipteran EcR, an Arthropod EcR, aHomopteran EcR and a Hemipteran EcR. More preferably, the EcR for use isa spruce budworm Choristoneura fumiferana EcR (“CfEcR”), a Tenebriomolitor EcR (“TmEcR”), a Manduca sexta EcR (“MsEcR”), a Heliothiesvirescens EcR (“HvEcR”), a silk moth Bombyx mori EcR (“BmEcR”), a fruitfly Drosophila melanogaster EcR (“DmEcR”), a mosquito Aedes aegypti EcR(“AaEcR”), a blowfly Lucilia capitata EcR (“LcEcR”), a Mediterraneanfruit fly Ceratitis capitata EcR (“CcEcR”), a locust Locusta migratoriaEcR (“ImEcR”), an aphid Myzus persicae EcR (“MpEcR”), a fiddler crab Ucapugilator EcR (“UpEcR”), or an ixodid tick Amblyomma americanum EcR(“AmaEcR”). Even more preferably, the LBD is from spruce budworm(Choristoneura fumiferana) EcR (“CfEcR”) or fruit fly Drosophilamelanogaster EcR (“DmEcR”).

[0174] Preferably, the LBD is from a truncated insect EcR. The insectEcR polypeptide truncation comprises a deletion of at least 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, or 265 amino acids. More preferably, theinsect EcR polypeptide truncation comprises a deletion of at least apartial polypeptide domain. Even more preferably, the insect EcRpolypeptide truncation comprises a deletion of at least an entirepolypeptide domain. In a specific embodiment, the insect EcR polypeptidetruncation comprises a deletion of at least an A/B-domain deletion, aC-domain deletion, a D-domain deletion, an E-domain deletion, anF-domain deletion, an A/B/C-domains deletion, an A/B1/2-C-domainsdeletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, anA/B/F-domains, and an A/B/C/F-domains deletion. A combination of severalcomplete and/or partial domain deletions may also be performed.

[0175] In a preferred embodiment, the ecdysone receptor ligand bindingdomain is encoded by a polynucleotide comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0176] In another preferred embodiment, the ecdysone receptor ligandbinding domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, and SEQ ID NO: 20.

[0177] Preferably, the RXR polypeptide is a mouse Mus musculus RXR(“MmRXR”) or a human Homo sapiens RXR (“HsRXR”). The RXR polypeptide maybe an RXR_(α), RXR_(β), or RXR_(γ) isoform.

[0178] Preferably, the LBD is from a truncated RXR. The RXR polypeptidetruncation comprises a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, or 265 amino acids. More preferably, the RXR polypeptidetruncation comprises a deletion of at least a partial polypeptidedomain. Even more preferably, the RXR polypeptide truncation comprises adeletion of at least an entire polypeptide domain. In a specificembodiment, the RXR polypeptide truncation comprises a deletion of atleast an A/B-domain deletion, a C-domain deletion, a D-domain deletion,an E-domain deletion, an F-domain deletion, an A/B/C-domains deletion,an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, anA/B/C/D/F-domains deletion, an A/B/F-domains, and an A/B/C/F-domainsdeletion A combination of several complete and/or partial domaindeletions may also be performed.

[0179] In a preferred embodiment, the retinoid X receptor ligand bindingdomain is encoded by a polynucleotide comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

[0180] In another preferred embodiment, the retinoid X receptor ligandbinding domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO: 40.

[0181] In a preferred embodiment, the gene expression cassette encodes ahybrid polypeptide comprising a DNA-binding domain encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of a GAL4 DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO:43) and an ecdysone receptor ligand binding domain encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,and SEQ ID NO: 10.

[0182] In another preferred embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a DNA-binding domain comprisinga polypeptide sequence selected from the group consisting of a GAL4 DBD(SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and an ecdysone receptorligand binding domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, and SEQ ID NO: 20.

[0183] In another preferred embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a DNA-binding domain encoded bya polynucleotide comprising a nucleic acid sequence selected from thegroup consisting of a GAL4 DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO:43) and a retinoid X receptor ligand binding domain encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO: 30.

[0184] In another preferred embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a DNA-binding domain comprisinga polypeptide sequence selected from the group consisting of a GALA DBD(SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and a retinoid X receptorligand binding domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, and SEQ ID NO:40.

[0185] In another preferred embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a transactivation domain encodedby a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45and an ecdysone receptor ligand binding domain encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9,and SEQ ID NO: 10.

[0186] In another preferred embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a transactivation domaincomprising a polypeptide sequence of SEQ ID NO: 46 and an ecdysonereceptor ligand binding domain comprising a polypeptide sequenceselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0187] In another preferred embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a transactivation domain encodedby a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45and a retinoid X receptor ligand binding domain encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO: 30.

[0188] In another preferred embodiment, the gene expression cassetteencodes a hybrid polypeptide comprising a transactivation domaincomprising a polypeptide sequence of SEQ ID NO: 46 and a retinoid Xreceptor ligand binding domain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0189] For purposes of this invention EcR and RXR also include syntheticand chimeric EcR and RXR and their homologs.

[0190] Polynucleotides of the Invention

[0191] The novel ecdysone receptor-based inducible gene expressionsystem of the invention comprises a gene expression cassette comprisinga polynucleotide that encodes a truncated EcR or RXR polypeptidecomprising a truncation mutation and is useful in methods of modulatingthe expression of a gene within a host cell.

[0192] Thus, the present invention also relates to a polynucleotide thatencodes an EcR or RXR polypeptide comprising a truncation mutationSpecifically, the present invention relates to an isolatedpolynucleotide encoding an EcR or RXR polypeptide comprising atruncation mutation that affects ligand binding activity or ligandsensitivity.

[0193] Preferably, the truncation mutation results in a polynucleotidethat encodes a truncated EcR polypeptide or a truncated RXR polypeptidecomprising a deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or265 amino acids. More preferably, the EcR or RXR polypeptide truncationcomprises a deletion of at least a partial polypeptide domain Even morepreferably, the EcR or RXR polypeptide truncation comprises a deletionof at least an entire polypeptide domain. In a specific embodiment, theEcR or RXR polypeptide truncation comprises a deletion of at least anA/B-domain deletion, a C-domain deletion, a D-domain deletion, anE-domain deletion, an F-domain deletion, an A/B/C-domains deletion, anA/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, anA/B/C/D/Fdomains deletion, an A/B/F-domains, and an A/B/C/F-domainsdeletion. A combination of several complete and/or partial domaindeletions may also be performed.

[0194] In a specific embodiment, the EcR polynucleotide according to theinvention comprises a polynucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,and SEQ ID NO: 10. In a specific embodiment, the polynucleotideaccording to the invention encodes a ecdysone receptor polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12 (CfEcR-1/2CDEF, whichcomprises the last 33 carboxy-terminal amino acids of C domain), SEQ IDNO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE),SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17 (DnEcR-1/2CDEF), SEQ ID NO: 18(DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), and SEQ ID NO: 20 (DmEc-DE).

[0195] In another specific embodiment, the RXR polynucleotide accordingto the invention comprises a polynucleotide sequence selected from thegroup consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 29, and SEQ ID NO: 30. In another specific embodiment, thepolynucleotide according to the invention encodes a truncated RXRpolypeptide comprising an amino acid sequence consisting of SEQ ID NO:31 (MmRXR-CDEF), SEQ ID NO: 32 (MmRXR-DEF), SEQ ID NO: 33 (MmRXR-EF),SEQ ID NO: 34 (MmRXR-truncatedEF), SEQ ID NO: 35 (MmRXR-E), SEQ ID NO:36 (HsRXR-CDEF), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF),SEQ ID NO: 39 (HSRXR-truncated EF), and SEQ ID NO: 40 (HsRXR-E).

[0196] In particular, the present invention relates to an isolatedpolynucleotide encoding an EcR or RXR polypeptide comprising atruncation mutation, wherein the mutation reduces ligand bindingactivity or ligand sensitivity of the EcR or RXR polypeptide. In aspecific embodiment, the present invention relates to an isolatedpolynucleotide encoding an ECR or RXR polypeptide comprising atruncation mutation that reduces steroid binding activity or steroidsensitivity of the EcR or RXR polypeptide. In a preferred embodiment,the present invention relates to an isolated polynucleotide encoding anEcR polypeptide comprising a truncation mutation that reduces steroidbinding activity or steroid sensitivity of the EcR polypeptide, whereinthe polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 3(CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 8 (DmEcR-DEF), or SEQID NO: 9 (DmEcR-EF). In another specific embodiment, the presentinvention relates to an isolated polynucleotide encoding an EcR or RXRpolypeptide comprising a truncation mutation that reduces non-steroidbinding activity or non-steroid sensitivity of the EcR or RXRpolypeptide. In a preferred embodiment, the present invention relates toan isolated polynucleotide encoding an EcR polypeptide comprising atruncation mutation that reduces non-steroid binding activity ornon-steroid sensitivity of the EcR polypeptide, wherein thepolynucleotide comprises a nucleic acid sequence of SEQ ID NO: 4(CfEcR-EF) or SEQ ID NO: 9 (DmEcR-EF).

[0197] The present invention also relates to an isolated polynucleotideencoding an EcR or RXR polypeptide comprising a truncation mutation,wherein the mutation enhances ligand binding activity or ligandsensitivity of the EcR or RXR polypeptide. In a specific embodiment, thepresent invention relates to an isolated polynucleotide encoding an EcRor RXR polypeptide comprising a truncation mutation that enhancessteroid binding activity or steroid sensitivity of the EcR or RXRpolypeptide. In another specific embodiment, the present inventionrelates to an isolated polynucleotide encoding an EcR or RXR polypeptidecomprising a truncation mutation that enhances non-steroid bindingactivity or non-steroid sensitivity of the EcR or RXR polypeptide. In apreferred embodiment, the present invention relates to an isolatedpolynucleotide encoding an EcR polypeptide comprising a truncationmutation that enhances non-steroid binding activity or non-steroidsensitivity of the EcR polypeptide, wherein the polynucleotide comprisesa nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF) or SEQ ID NO: 8(DmEcR-DEF).

[0198] The present invention also relates to an isolated polynucleotideencoding a retinoid X receptor polypeptide comprising a truncationmutation that increases ligand sensitivity of a heterodimer comprisingthe mutated retinoid X receptor polypeptide and a dimerization partner.Preferably, the isolated polynucleotide encoding a retinoid X receptorpolypeptide comprising a truncation mutation that increases ligandsensitivity of a heterodimer comprises a polynucleotide sequenceselected from the group consisting of SEQ ID NO: 23 (MmRXR-EF), SEQ IDNO: 24 (MmRXR-truncatedEF), SEQ ID NO: 28 (HsRXR-EF), or SEQ ID NO: 29(HsRXR-truncated EF). In a specific embodiment, the dimerization partneris an ecdysone receptor polypeptide. Preferably, the dimerizationpartner is a truncated EcR polypeptide. More preferably, thedimerization partner is an EcR polypeptide in which domains A/B/C havebeen deleted. Even more preferably, the dimerization partner is an EcRpolypeptide comprising an amino acid sequence of SEQ ID NO: 13(CfEcR-DEF) or SEQ ID NO: 18 (DmEcR-DEF).

[0199] Polypeptides of the Invention

[0200] The novel ecdysone receptor-based inducible gene expressionsystem of the invention comprises a polynucleotide that encodes atruncated EcR or RXR polypeptide and is useful in methods of modulatingthe expression of a gene within a host cell. Thus, the present inventionalso relates to an isolated truncated EcR or RXR polypeptide encoded bya polynucleotide or a gene expression cassette according to theinvention. Specifically, the present invention relates to an isolatedtruncated EcR or RXR polypeptide comprising a truncation mutation thataffects ligand binding activity or ligand sensitivity encoded by apolynucleotide according to the invention.

[0201] The present invention also relates to an isolated truncated EcRor RXR polypeptide comprising a truncation mutation. Specifically, thepresent invention relates to an isolated EcR or RXR polypeptidecomprising a truncation mutation that affects ligand binding activity orligand sensitivity.

[0202] Preferably, the truncation mutation results in a truncated EcRpolypeptide or a truncated RXR polypeptide comprising a deletion of atleast 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. Morepreferably, the EcR or RXR polypeptide truncation comprises a deletionof at least a partial polypeptide domain. Even more preferably, the EcRor RXR polypeptide truncation comprises a deletion of at least an entirepolypeptide domain. In a specific embodiment, the EcR or RXR polypeptidetruncation comprises a deletion of at least an A/B-domain deletion, aC-domain deletion, a D-domain deletion, an E-domain deletion, anF-domain deletion, an A/B/C-domains deletion, an A/B/1/2C-domainsdeletion, an A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, anA/B/F-domains, and an A/B/C/F-domains deletion. A combination of severalcomplete and/or partial domain deletions may also be performed.

[0203] In a preferred embodiment, the isolated truncated ecdysonereceptor polypeptide is encoded by a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ ID NO:1 (CfEcR-CDEF), SEQ ID NO: 2 (CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF),SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6(DnEcR-CDEF), SEQ ID NO: 7 (DmEcR-1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF),SEQ ID NO: 9 (DmEcR-EF), and SEQ ID NO: 10 (DmEcR-DE). In anotherpreferred embodiment, the isolated truncated ecdysone receptorpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12 (CfEcR-1/2CDEF),SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15(CfEcR-DE), SEQ ID NO: 16 (DnEcR-CDEF), SEQ ID NO: 17 (DmEcR-1/2CDEF),SEQ ID NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), and SEQ ID NO: 20(DmEcR-DE).

[0204] In a preferred embodiment, the isolated truncated RXR polypeptideis encoded by a polynucleotide comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO: 21 (MmRXR-CDEF), SEQ IDNO: 22 (MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24(MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26(HsRXR-CDEF), SEQ ID NO: 27.(HsRXR-DEF), SEQ ID NO: 28 (HsRXR-EF), SEQID NO: 29 (HsRXR-truncatedEF) and SEQ ID NO: 30 (HsRXR-E). In anotherpreferred embodiment, the isolated truncated RXR polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:31 (MmRXR-CDEF), SEQ ID NO: 32 (MmRXR-DEF), SEQ ID NO: 33 (RxR-EF), SEQID NO: 34 (MmRXR-truncatedEF), SEQ ID NO: 35 (RXR-E), SEQ ID NO: 36(HsRXR-CDEP), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF), SEQID NO: 39 (HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).

[0205] The present invention relates to an isolated EcR or RXRpolypeptide comprising a truncation mutation that reduces ligand bindingactivity or ligand sensitivity of the EcR or RXR polypeptide, whereinthe polypeptide is encoded by a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ ID NO:1 (CfEcR-CDEF), SEQ ID NO: 2 (CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF),SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6(DmEcR-CDEF), SEQ ID NO: 7 (DmEcR1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF), SEQID NO: 9 (DmEcR-EF), SEQ ID NO: 10 (DmEcR-DE), SEQ ID NO: 21(MmRXR-CDEF), SEQ ID NO: 22 (MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26(HsRXR-CDEF), SEQ ID NO: 27 (HsRXR-DEF), SEQ ID NO: 28 (HsRXR-EF), SEQID NO: 29 (HsRXR-truncatedEF), and SEQ ID NO: 30 (HsRXR-E).

[0206] Thus, the present invention relates to an isolated truncated EcRor RXR polypeptide comprising a truncation mutation that reduces ligandbinding activity or ligand sensitivity of the EcR or RXR polypeptide,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12(CfEcR-1/2CDEF), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF),SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17(DmEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF),SEQ ID NO: 20 (DmEcR10 DE), SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32(MmRXR-DEF), SEQ ID NO: 33 (MmR-EF), SEQ ID NO: 34 (MmRXR-truncatedEF),SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36 (HsRXR-CDEF), SEQ ID NO: 37(HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF), SEQ ID NO: 39(HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).

[0207] In a specific embodiment, the present invention relates to anisolated EcR or RXR polypeptide comprising a truncation mutation thatreduces steroid binding activity or steroid sensitivity of the EcR orRXR polypeptide. In a preferred embodiment, the present inventionrelates to an isolated EcR polypeptide comprising a truncation mutationthat reduces steroid binding activity or steroid sensitivity of the EcRpolypeptide, wherein the EcR polypeptide is encoded by a polynucleotidecomprising a nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF), SEQ IDNO: 4 (CfEcR-EF), SEQ ID NO: 8 (DmEcR-DEF), or SEQ ID NO: 9 (DmEcR-EF).Accordingly, the present invention also relates to an isolated truncatedEcR or RXR polypeptide comprising a truncation nmtation that reducessteroid binding activity or steroid sensitivity of the EcR or RXRpolypeptide. In a preferred embodiment, the present invention relates toan isolated EcR polypeptide comprising a truncation mutation thatreduces steroid binding activity or steroid sensitivity of the EcRpolypeptide, wherein the EcR polypeptide comprises an amino acidsequence of SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ IDNO: 18 (DmEcR-DEF), or SEQ ID NO: 19 (DmEcR-EF).

[0208] In another specific embodiment, the present invention relates toan isolated EcR or RXR polypeptide comprising a truncation mutation thatreduces non-steroid binding activity or non-steroid sensitivity of theEcR or RXR polypeptide. In a preferred embodiment, the present inventionrelates to an isolated EcR polypeptide comprising a truncation mutationthat reduces non-steroid binding activity or non-steroid sensitivity ofthe EcR polypeptide, wherein the EcR polypeptide is encoded by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 4(CfEcR-EF) or SEQ ID NO: 9 (DmEcR-EF). Accordingly, the presentinvention also relates to an isolated truncated EcR or RXR polypeptidecomprising a truncation mutation that reduces non-steroid bindingactivity or steroid sensitivity of the EcR or RXR polypeptide. In apreferred embodiment, the present invention relates to an isolated EcRpolypeptide comprising a truncation mutation that reduces non-steroidbinding activity or non-steroid sensitivity of the EcR polypeptide,wherein the EcR polypeptide comprises an amino acid sequence of SEQ IDNO: 14 (CfEcR-EF) or SEQ ID NO: 19 (DmEcR-EF).

[0209] In particular, the present invention relates to an isolated EcRor RXR polypeptide comprising a truncation mutation that enhances ligandbinding activity or ligand sensitivity of the EcR or RXR polypeptide,wherein the polypeptide is encoded by a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ ID NO:1 (CfEcR-CDEF), SEQ ID NO: 2 (CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF),SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6(DmEcR-CDEF), SEQ ID NO: 7 (DmEcR-1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF),SEQ ID NO: 9 (DmEcR-EF), SEQ ID NO: 10 (DmEcR-DE), SEQ ID NO: 21(MmRXR-CDEP), SEQ ID NO: 22 (MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26(HsRXR-CDEF), SEQ ID NO: 27 (HsRXRDEF), SEQ ID NO: 28 (HsRXR-EF), SEQ IDNO: 29 (HsRXR-truncated EF), and SEQ ID NO: 30 (HsRXR-E).

[0210] The present invention relates to an isolated EcR or RXRpolypeptide comprising a truncation mutation that enhances ligandbinding activity or ligand sensitivity of the EcR or RXR polypeptide,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12(CfEcR-1/2CDEF), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF),SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17(DmEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF),SEQ ID NO: 20 (DmEcR-DE), SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32(MmRXR-DEF), SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34 (MmRXR-runcatedEF),SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36 (HsRXR-CDEF), SEQ ID NO: 37(HsRXR-DEF), SEQ ID NO: 39 (HsRXR-EF), SEQ ID NO: 39(HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).

[0211] The present invention relates to an isolated EcR or RXRpolypeptide comprising a truncation mutation that enhances ligandbinding activity or ligand sensitivity of the EcR or RXR polypeptide. Ina specific embodiment, the present invention relates to an isolated EcRor RXR polypeptide comprising a truncation mutation that enhancessteroid binding activity or steroid sensitivity of the EcR or RXRpolypeptide. Accordingly, the present invention also relates to anisolated EcR or RXR polypeptide comprising a truncation mutation thatenhances steroid binding activity or steroid sensitivity of the EcR orRXR polypeptide.

[0212] In another specific embodiment, the present invention relates toan isolated EcR or RXR polypeptide comprising a truncation mutation thatenhances non-steroid binding activity or non-steroid sensitivity of theEcR or RXR polypeptide. In a preferred embodiment, the present inventionrelates to an isolated EcR polypeptide comprising a truncation mutationthat enhances non-steroid binding activity or non-steroid sensitivity ofthe EcR polypeptide, wherein the EcR polypeptide is encoded by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 3(CfEcR-DEF) or SEQ ID NO: 8 (DmEcR-DEF). Accordingly, the presentinvention also relates to an isolated EcR or RXR polypeptide comprisinga truncation mutation that enhances non-steroid binding activity orsteroid sensitivity of the EcR or RXR polypeptide. In a preferredembodiment, the present invention relates to an isolated EcR polypeptidecomprising a truncation mutation that enhances non-steroid bindingactivity or non-steroid sensitivity of the EcR polypeptide, wherein theEcR polynucleotide comprises an amino acid sequence of SEQ ID NO: 13(CfEcR-DEF) or SEQ ID NO: 18 (DmEcR-DEF).

[0213] The present invention also relates to an isolated retinoid Xreceptor polypeptide comprising a truncation mutation that increasesligand sensitivity of a heterodimer comprising the mutated retinoid Xreceptor polypeptide and a dimerization partner. Preferably, theisolated retinoid X receptor polypeptide comprising a truncationmutation that increases ligand sensitivity of a heterodimer is encodedby a polynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24(MmRXR-truncatedEF), SEQ ID NO: 28 (HsRXR-EF), or SEQ ID NO: 29(HsRXR-truncatedEF). More preferably, the isolated polynucleotideencoding a retinoid X receptor polypeptide comprising a truncationmutation that increases ligand sensitivity of a heterodimer comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 33(MnRXR-EF), SEQ ID NO: 34 (nRXR-trancatedEF), SEQ ID NO: 38 (HsRXR-EF),or SEQ ID NO: 39 (HsRXR-truncatedEF).

[0214] In a specific embodiment, the dimerization partner is an ecdysonereceptor polypeptide. Preferably, the dimerization partner is atruncated EcR polypeptide. More preferably, the dimerization partner isan EcR polypeptide in which domains A/B/C have been deleted. Even morepreferably, the dimerization partner is an EcR polypeptide comprising anamino acid sequence of SEQ ID NO: 13 (CfEcR-DEF) or SEQ ID NO: 18(DmEcR-DEF).

[0215] Method of Modulating Gene Expression of the Invention

[0216] Applicants' invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation systemaccording to the invention. Specifically, Applicants' invention providesa method of modulating the expression of a gene in a host cellcomprising the steps of: a) introducing into the host cell a geneexpression modulation system according to the invention; and b)introducing into the host cell a ligand that independently combines withthe ligand binding domains of the first polypeptide and the secondpolypeptide of the gene expression modulation system, wherein the geneto be expressed is a component of a gene expression cassette comprising:i) a response element comprising a domain to which the DNA bindingdomain of the first polypeptide binds; ii) a promoter that is activatedby the transactivation domain of the second polypeptide; and iii) a genewhose expression is to be modulated, whereby a complex is formedcomprising the ligand, the first polypeptide of the gene expressionmodulation system and the second polypeptide of the gene expressionmodulation system, and whereby the complex modulates expression of thegene in the host cell.

[0217] Genes of interest for expression in a host cell using Applicants'methods may be endogenous genes or heterologous genes. Nucleic acid oramino acid sequence information for a desired gene or protein can belocated in one of many public access databases, for example, GENBANK,EMBL, Swiss-Prot, and PIR, or in many biology related journalpublications. Thus, those skilled in the art have access to nucleic acidsequence information for virtually all known genes. Such information canthen be used to construct the desired constructs for the insertion ofthe gene of interest within the gene expression cassettes used inApplicants' methods described herein.

[0218] Examples of genes of interest for expression in a host cell usingApplicants' methods include, but are not limited to: antigens producedin plants as vaccines, enzymes like alphaamylase, phytase, glucanes, andxylanse, genes for resistance against insects, nematodes, fungi,bacteria, viruses, and abiotic stresses, nutraceuticals,pharmaceuticals, vitamins, genes for modifying amino acid content,herbicide resistance, cold, drought, and heat tolerance, industrialproducts, oils, protein, carbohydrates, antioxidants, male sterileplants, flowers, fuels, other output traits, genes encodingtherapeutically desirable polypeptides or products, such as genes thatcan provide, modulate, alleviate, correct and/or restore polypeptidesimportant in treating a condition, a disease, a disorder, a dysfunction,a genetic defect, and the like.

[0219] Acceptable ligands are any that modulate expression of the genewhen binding of the DNA binding domain of the two hybrid system to theresponse element in the presence of the ligand results in activation orsuppression of expression of the genes. Preferred ligands includeponasterone, muristerone A, N,N′-diacylhydrazines such as thosedisclosed in U.S. Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and5,378,726; dibenzoylalkyl cyanohydrazines such as those disclosed inEuropean Application No. 461,809; N-alkyl-N,N′-diaroylhydrazines such asthose disclosed in U.S. Pat. No. 5,225,443;N-acyl-N-alkylarbonylhydrazines such as those disclosed in EuropeanApplication No. 234,994; N-aroyl-N-alkyl-N′-aroylhydrazines such asthose described in U.S. Pat. No. 4,985,461; each of which isincorporated herein by reference and other similar materials including3,5-di-tert-butyl-4-hydroxy-N-isobutylbenzamide, 8-O-acetylharpagide,and the like.

[0220] Preferably, the ligand for use in Applicants' method ofmodulating expression of gene is a compound of the formula:

[0221] wherein:

[0222] E is a (C₄-C₆)alkyl containing a tertiary carbon or acyano(C₃-Cs)alkyl containing a tertiary carbon;

[0223] R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂, CH2F, CH₂Cl,CH₂OH, CH₂OMe, CH₂CN, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe,OEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, SCN, or SCHF₂;

[0224] R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F,CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C°CH, 1-propynyl, 2-propynyl, vinyl,Ac, F, Cl, OH, OMe, OEt, O-nPr, OAc, NMe₂, NEt₂, SMe, SEt, SOCF₃,OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido, OCF3, OCHF₂,O-i-Pr, SCN, SCHF₂, SOMe, NH—CN, or joined with R³ and the phenylcarbons to which R² and R³ are attached to form an ethylenedioxy, adihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or adihydropyryl ring with the oxygen adjacent to a phenyl carbon;

[0225] R³ is H, Et, or joined with R² and the phenyl carbons to which R²and R³ are attached to form an ethylenedioxy, a dihydrofuryl ring withthe oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with theoxygen adjacent to a phenyl carbon;

[0226] R⁴, R⁵, and R⁶ are independently H, Me, Et, F, Cl, Br, formyl,CF₃, CHF₂, CHCl₂, CH2F, CH₂Cl, CH₂OH, CN, C°CH, 1-propynyl, 2-propynyl,vinyl, OMe, OEt, SMe, or SEt.

[0227] Applicants' invention provides for modulation of gene expressionin prokaryotic and eukaryotic host cells. Thus, the present inventionalso relates to a method for modulating gene expression in a host cellselected from the group consisting of a bacterial cell, a fungal cell, ayeast cell, a plant cell, an animal cell, and a mamalian cell.Preferably, the host cell is a yeast cell, a plant cell, a murine cell,or a human cell.

[0228] Expression in transgenic host cells may be useful for theexpression of various polypeptides of interest including but not limitedto therapeutic polypeptides, pathway intermediates; for the modulationof pathways already existing in the host for the synthesis of newproducts heretofore not possible using the host; cell based assays; andthe like. Additionally the gene products may be useful for conferringhigher growth yields of the host or for enabling alternative growth modeto is utilized.

[0229] Host Cells and Non-Human Organisms of the Inveniion

[0230] As described above, the gene expression modulation system of thepresent invention may be used to modulate gene expression in a hostcell. Expression in transgenic host cells may be useful for theexpression of various genes of interest. Thus, Applicants' inventionalso provides an isolated host cell comprising a gene expression systemaccording to the invention. The present invention also provides anisolated host cell comprising a gene expression cassette according tothe invention. Applicants' invention also provides an isolated host cellcomprising a polynucleotide or polypeptide according to the inventionThe isolated host cell may be either a prokaryotic or a eukaryotic hostcell.

[0231] Preferably, the host cell is selected from the group consistingof a bacterial cell, a fungal cell, a yeast cell, a plant cell, ananimal cell, and a mammalian cell. Examples of preferred host cellsinclude, but are not limited to, fungal or yeast species such asAspergillus, Trichoderna, Saccharomyces, Pichia, Candida, Hansenula, orbacterial species such as those in the genera Synechocystis,Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus,Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter,Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacteriuinand Klebsiella, plant, animal, and mammalian host cells. Morepreferably, the host cell is a yeast cell, a plant cell, a murine cell,or a human cell.

[0232] In a specific embodiment, the host cell is a yeast cell selectedfrom the group consisting of a Saccharomyces, a Pichia, and a Candidahost cell.

[0233] In another specific embodiment, the host cell is a plant cellselected from the group consisting of an apple, Arabidopsis, bajra,banana, barley, bean, beet, blackgram, chickpea, chili, cucumber,eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum,papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato,pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheathost cell.

[0234] In another specific embodiment, the host cell is a murine cell.

[0235] In another specific embodiment, the host cell is a human cell.

[0236] Host cell transformation is well known in the art and may beachieved by a variety of methods including but not limited toelectroporation, viral infection, plasmid/vector transfection, non-viralvector mediated transfection, Agrobacterium-mediated transformation,particle bombardment, and the like. Expression of desired gene productsinvolves culturing the transformed host cells under suitable conditionsand inducing expression of the transformed gene. Culture conditions andgene expression protocols in prokaryotic and eukaryotic cells are wellknown in the art (see General Methods section of Examples). Cells may beharvested and the gene products isolated according to protocols specificfor the gene product.

[0237] In addition, a host cell may be chosen which modulates theexpression of the inserted polynucleotide, or modifies and processes thepolypeptide product in the specific fashion desired. Different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification (e.g., glycosylation,cleavage [e.g., of signal sequence]) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a non-glycosylated coreprotein product. However, a polypeptide expressed in bacteria may not beproperly folded. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the polypeptide's activity. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent.

[0238] Applicants' invention also relates to a non-human organismcomprising an isolated host cell according to the invention. Preferably,the non-human organism is selected from the group consisting of abacterium, a fungus, a yeast, a plant, an animal, and a mammal. Morepreferably, the non-human organism is a yeast, a plant, a mouse, a rat,a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, amonkey, or a chimpanzee.

[0239] In a specific embodiment, the non-human organism is a yeastselected from the group consisting of Saccharomyces, Pichia, andCandida.

[0240] In another specific embodiment, the non-human organism is a plantselected from the group consisting of an apple, Arabidopsis, bajra,banana, barley, beans, beet, blackgram, chickpea, chili, cucumber,eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum,papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato,pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.

[0241] In another specific embodiment, the non-human organism is a Musmusculus mouse.

[0242] Measuring Gene Expression/Transcription

[0243] One useful measurement of Applicants' methods of the invention isthat of the transcriptional state of the cell including the identitiesand abundances of RNA, preferably mRNA species. Such measurements areconveniently conducted by measuring cDNA abundances by any of severalexisting gene expression technologies.

[0244] Nucleic acid array technology is a useful technique fordetermining differential mRNA expression Such technology includes, forexample, oligonucleotide chips and DNA microarrays. These techniquesrely on DNA fragments or oligonucleotides which correspond to differentgenes or cDNAs which are immobilized on a solid support and hybridizedto probes prepared from total mRNA pools extracted from cells, tissues,or whole organisms and converted to cDNA. Oligonucleotide chips arearrays of oligonucleotides synthesized on a substrate usingphotolithographic techniques. Chips have been produced which can analyzefor up to 1700 genes. DNA microarrays are arrays of DNA samples,typically PCR products, that are robotically printed onto a microscopeslide. Each gene is analyzed by a full or partial-length target DNAsequence. Microarrays with up to 10,000 genes are now routinely preparedcommercially. The primary difference between these two techniques isthat oligonucleotide chips typically utilize 25-mer oligonucleotideswhich allow fractionation of short DNA molecules whereas the larger DNAtargets of microarrays, approximately 1000 base pairs, may provide moresensitivity in fractionating complex DNA mixtures.

[0245] Another useful measurement of Applicants' methods of theinvention is that of determining the translation state of the cell bymeasuring the abundances of the constituent protein species present inthe cell using processes well known in the art.

[0246] Where identification of genes associated with variousphysiological functions is desired, an assay may be employed in whichchanges in such functions as cell growth, apoptosis, senescence,differentiation, adhesion, binding to a specific molecules, binding toanother cell, cellular organization, organogenesis, intracellulartransport, transport facilitation, energy conversion, metabolism,myogenesis, neurogenesis, and/or hematopoiesis is measured.

[0247] In addition, selectable marker or reporter gene expression may beused to measure gene expression modulation using Applicants' invention.

[0248] Other methods to detect the products of gene expression are wellknown in the art and include Southern blots (DNA detection), dot or slotblots (DNA, RNA), Northem blots (RNA), and RT-PCR(RNA) analyses.Although less preferred, labeled proteins can be used to detect aparticular nucleic acid sequence to which it hybidizes.

[0249] In some cases it is necessary to amplify the amount of a nucleicacid sequence. This may be carried out using one or more of a number ofsuitable methods including, for example, polymerase chain reaction(“PCR”), ligase chain reaction (“LCR”), strand displacementamplification (“SDA”), transcription-based amplification, and the like.PCR is carried out in accordance with known techniques in which, forexample, a nucleic acid sample is treated in the presence of a heatstable DNA polymerase, under hybridizing conditions, with oneoligonucleotide primer for each strand of the specific sequence to bedetected. An extension product of each primer that is synthesized iscomplementary to each of the two nucleic acid strands, with the primerssufficiently complementary to each strand of the specific sequence tohybridize therewith. The extension product synthesized from each primercan also serve as a template for further synthesis of extension productsusing the same pmers. Following a sufficient number of rounds ofsynthesis of extension products, the sample may be analyzed as describedabove to assess whether the sequence or sequences to be detected arepresent

[0250] The present invention may be better understood by reference tothe following non-limiting Examples, which are provided as exemplary ofthe invention.

EXAMPLES

[0251] General Methods

[0252] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Assoc. andWiley-Interscience (1987).

[0253] Methods for plant tissue culture, transformation, plant molecularbiology, and plant, general molecular biology may be found in PlantTissue Culture Concepts and Laboratory Exercises edited by RN Trigianoand DJ Gray, 2^(nd) edition, 2000, CRC press, New York; AgrobacteriumProtocols edited by KMA Gartland and MR Davey, 1995, Humana Press,Totowa, N.J.; Methods in Plant Molecular Biology, P. Maliga et al.,1995, Cold Spring Harbor Lab Press, New York; and Molecular Cloning, J.Sambrook et al., 1989, Cold Spring Harbor Lab Press, New York.

[0254] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of host cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St Louis, Mo.) unless otherwise specified.

[0255] Manipulations of genetic sequences may be accomplished using thesuite of programs available from the Genetics Computer Group Inc.(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.). Where the GCG program “Pileup” is used the gap creation defaultvalue of 12, and the gap extension default value of 4 may be used. Wherethe CGC “Gap” or “Bestfit” programs is used the default gap creationpenalty of 50 and the default gap extension penalty of 3 may be used. Inany case where GCG program parameters are not prompted for, in these orany other GCG program, default values may be used.

[0256] The meaning of abbreviations is as follows: “1” means hour(s),“min” means minute(s), “sec” means second(s), “d” means day(s), “μl”means microliter(s), “ml” means milliliter(s), “L” means liter(s), “μM”means micromolar, “mM” means millimolar, “μg” means microgram(s), “mg”means milligram(s), “A” means adenine or adenosine, “T” means thymine orthymidine, “G” means guanine or guanosine, “C” means cytidine orcytosine, “x g” means times gravity, “nt” means nucleotide(s), “aa”means amino acid(s), “bp” means base pair(s), “kb” means kilobase(s),“k” means kilo, “μ” means micro, and “° C.” means degrees Celsius.

Example 1

[0257] Applicants' improved EcR-based inducible gene modulation systemwas developed for use in various applications including gene therapy,expression of proteins of interest in host cells, production oftransgenic organisms, and cell-based assays. This Example describes theconstruction and evaluation of several gene expression cassettes for usein the EcR-based inducible gene expression system of the invention.

[0258] In various cellular backgrounds, including mammalian cells,insect ecdysone receptor (EcR) heterodimerizes with retinoid X receptor(RXR) and, upon binding of ligand, transactivates genes under thecontrol of ecdysone response elements. Applicants constructed severalEcR-based gene expression cassettes based on the spruce budwormChoristoneura fumiferana EcR (“CfEcR”; full length polynucleotide andamino acid sequences are set forth in SEQ ID NO: 49 and SEQ ID NO: 50,respectively), C. fumiferana ultraspiracle (“CfUSP”; full lengthpolynucleotide and amino acid sequences are set forth in SEQ ID NO: 51and SEQ ID NO: 52, respectively), and mouse Mus musculus RXRA (MmRXRα;full length polynucleotide and amino acid sequences are set forth in SEQID NO: 53 and SEQ ID NO: 54, respectively). The prepared receptorconstructs comprise a ligand binding domain of EcR and of RXR or of USP;a DNA binding domain of GAL4 or of EcR; and an activation domain ofVP16. The reporter constructs include a reporter gene, luciferase orLacZ, operably linked to a synthetic promoter construct that compriseseither GAIA or EcR/USP binding sites (response elements). Variouscombinations of these receptor and reporter constructs werecotransfected into CHO, NIH3T3, CV1 and 293 cells. Gene inductionpotential (magnitude of induction) and ligand specificity andsensitivity were examined using four different ligands: two steroidalligands (ponasterone A and muristerone A) and two non-steroidal ligands(N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazineandN-(3,4(1,2-ethylenedioxy)-2-methylbenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine)in a dose-dependent induction of reporter gene expression in thetransfected cells. Reporter gene expression activities were assayed at24 hr or 48 hr after ligand addition.

[0259] Gene Expression Cassettes: Ecdysone receptor-based, chemicallyinducible gene expression cassettes (switches) were constructed asfollowed, using standard cloning methods available in the art. Thefollowing is brief description of preparation and compositionof eachswitch.

[0260] 11.1—GALAEcRNP16RXR: The D, E, and F domains from spruce budwormChoristoneura fumiferana EcR (“CfEcRDEF”; SEQ ID NO: 3) were fused toGAL4 DNA binding domain (“DNABD”; SEQ ID NO: 41) and placed under thecontrol of an SV40e promoter (SEQ ID NO: 55). The DEF domains from mouse(Mus musculus) RXR (“MmRXRDEF”; SEQ ID NO: 22) were fused to theactivation domain from VP16 (“VP16AD”; SEQ ID NO: 45) and placed underthe control of an SV40e promoter (SEQ ID NO: 55). Five consensus GAL4binding sites (“5XGALARE”; comprising 5, GAL4RE comprising SEQ ID NO:47) were fused to a synthetic E1b minimal promoter (SEQ ID NO: 56) andplaced upstream of the luciferase gene (SEQ ID NO: 57).

[0261] 1.2—GALAEcR/VP16USP: This construct was prepared in the same wayas in switch 1.1 above except MmRXRDEF was replaced with the D, E and Fdomains from spruce budworm USP (“CfUSPDEF”; SEQ ID NO: 58). Theconstructs used in this example are similar to those disclosed in U.S.Pat. No. 5,880,333 except that Choristoneura fumiferana USP rather thanDrosophila melanogaster USP was utilized.

[0262] 1.3—GAIARXR/VP16CfEcR: MmRXRDEF (SEQ ID NO: 22) was fused to aGAL4DNABD (SEQ ID NO: 41) and CfEcRCDEF (SEQ ID NO: 1) was fused to aVP16AD (SEQ ID NO: 45).

[0263] 1.4—GAL4RXR/VP16DmEcR: This construct was prepared in the sameway as switch 1.3 except CfEcRCDEF was replaced with DmEcRCDEF (SEQ IDNO: 6).

[0264] 1.5—GAl4USP/VP16CfEcR: This construct was prepared in the sameway as switch 1.3 except MmRXRDEF was replaced with CFUSPDEF (SEQ ID NO:58).

[0265] 1.6—GALARXRCfEcRVP16: This construct was prepared so that boththe GAL4 DNABD and the VP16AD were placed on the same molecule.GAL4DNABD (SEQ ID NO: 41) and VP16AD (SEQ ID NO: 45) were fused toCfEcRDEF (SEQ ID NO: 3) at N-and C-termini respectively. The fusion wasplaced under the control of an SV40e promoter (SEQ ID NO: 55).

[0266] 1.7—VP16CfEcR: This construct was prepared such that CfEcRCDEF(SEQ ID NO: 1) was fused to VP16AD (SEQ ID NO: 45) and placed under thecontrol of an SV40e promoter (SEQ ID NO: 55). Six ecdysone responseelements (“EcRE”; SEQ ID NO: 59) from the hsp27 gene were placedupstream of the promoter and a luciferase gene (SEQ ID NO: 57). Thisswitch most probably uses endogenous RXR.

[0267] 1.8—DmVgRXR: This system was purchased from Invitrogen Corp.,Carlsbad, Calif. It comprises a Drosophila melanogaster EcR (“DnEcR”)with a modified DNABD fused to VP16AD and placed under the control of aCMV promoter (SEQ D) NO: 60). Full length MmRXR (SEQ ID NO: 53) wasplaced under the control of the RSV promoter (SEQ ID NO: 61). Thereporter, pIND(SP1)LacZ, contains five copies of a modified ecdysoneresponse element (“EcRE”, E/GRE), three copies of an SP1 enhancer, and aminimal heat shock promoter, all of which were placed upstream to theLacZ reporter gene.

[0268] 1.9—CfVgRXR: This example was prepared in the same way as switch1.8 except DmEcR was replaced with a truncated CfER comprising a partialA/B domain and the complete CDEF domains [SEQ ID NO: 62 (polynucleotide)and SEQ ID NO: 63 (polypeptide)].

[0269] 1.10—CfVgRXRdel: This example was prepared in the same way asswitch 1.9 except MmRXR (SEQ ID NO: 53) was deleted.

[0270] Cell lines: Four cell lines: CHO, Chinese hamster Cricetulusgriseus ovarian cell line; NIH3T3 (3T3) mouse Mus musculus cell line;293 human Homo sapiens kidney cell line, and CV1 Afican green monkeykidney cell line were used in these experiments. Cells were maintainedin their respective media and were subcultured when they reached 60%confluency. Standard methods for culture and maintenance of the cellswere followed.

[0271] Transfections: Several commercially available lipofactors as wellas electroporation methods were evaluated and the best conditions fortransfection of each cell line were developed. CHO, NIH3T3, 293 and CV1cells were grown to 60% confluency. DNAs corresponding to the variousswitch constructs outlined in Examples 1.1 through 1.10 were transfectedinto CHO cells, NIH3T3 cells, 293 cells, or CV1 cells as follows.

[0272] CHO cells: Cells were harvested when they reach 60-80% confluencyand plated in 6- or 12 or 24-well plates at 250,000, 100,000, or 50,000cells in 2.5, 1.0, or 0.5 ml of growth medium containing 10% Fetalbovine serum respectively. The next day, the cells were rinsed withgrowth medium and transfected for four hours. LipofectAMINE™ 2000 (LifeTechnologies Inc,) was found to be the best transfection reagent forthese cells. For 12-well plates, 4 μl of LipofectAMINEm 2000 was mixedwith 100 μl of growth medium 1.0 μg of reporter construct and 0.25 μg ofreceptor construct(s) were added to the transfection mix. A secondreporter construct was added [pTKRL (Promega), 0.1 μg/transfection mix]and comprised a Renilla luciferase gene (SEQ ID NO: 64) operably linkedand placed under the control of a thyrnidine kinase (IK) constitutivepromoter and was used for normalization. The contents of thetransfection mix were mixed in a vortex mixer and let stand at roomtemperature for 30 min. At the end of incubation, the transfection mixwas added to the cells maintained in 400 μl growth medium. The cellswere maintained at 37° C. and 5% CO₂ for four hours. At the end ofincubation, 500 μl of growth medium containing 20% FBS and either DMSO(control) or a DMSO solution of appropriate ligands were added and thecells were maintained at 37° C. and 5% CO₂ for 24-48 hr. The cells wereharvested and reporter activity was assayed. The same procedure wasfollowed for 6 and 24 well plates as well except all the reagents weredoubled for 6 well plates and reduced to half for 24-well plates.

[0273] NIH3T3 Cells: Superfect™ (Qiagen Inc.) was found to be the besttrasfection reagent for 3T3 cells. The same procedures described for CHOcells were followed for 3T3 cells as well with two modifications. Thecells were plated when they reached 50% confluency. 125,000 or 50,000 or25,000 cells were plated per well of 6 or 12- or 24-well platesrespectively. The GA14EcR/VP16RXR and reporter vector DNAs weretransfected into NIH3T3 cells, the transfected cells were grown inmedium containing PonA, MurA,N-(2-ethyl-3methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-t-butylhydrazine,orN-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N′-(3,5-dimethybenzoyl)-N′-tert-butylhydrazinefor 48 hr. The ligand treatments were performed as described in the CHOcell section above.

[0274] 293 Cells: LipofectAMINE™ 2000 (Life Technologies) was found tobe the best lipofactor for 293 cells. The same procedures described forCHO were followed for 293 cells except that the cells were plated inbiocoated plates to avoid clumping. The ligand treatments were performedas described in the CHO cell section above.

[0275] CV1 Cells: LipofectAMiNE™ plus (Life Technologies) was found tobe the best lipofactor for CV1 cells. The same procedures described forNIH3T3 cells were followed for CV1 cells

[0276] Ligands: Ponasterone A and Muristerone A were purchased fromSigma Chemical Company. The two non-steroidsN-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-t-butylhydrazine,orN-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazineare synthetic stable ecdysteroids synthesized at Rohm and Haas Company.All ligands were dissolved in DMSO and the final concentration of DMSOwas maintained at 0.1% in both controls and treatments.

[0277] Reporter Assays: Cells were harvested 24-48 hr after addingligands. 0.125, 250, or 500 μl of passive lysis buffer (part ofDual-luciferase™ reporter assay system from Promega Corporation) wereadded to each well of 24- or 12- or 24well plates respectively. Theplates were placed on a rotary shaker for 15 min. Twenty μl of lysatewas assayed. Luciferase activity was measured using Dual-luciferase™reporter assay system from Promega Corporation following themanufacturer's instructions. β-Galactosidase was measured usingGalacto-Star™ assay kit from TROPIX following the manufacturer'sinstructions. All luciferase and β-galactosidase activities werenormalized using Renilla luciferase as a standard. Fold activities werecalculated by dividing normalized relative light units (“RLU”) in ligandtreated cells with normlized RLU in DMSO treated cells (untreatedcontrol).

[0278] The results of these experiments are provided in the followingtables. TABLE 1 Transactivation of reporter genes through variousswitches in CHO cells Mean Fold Activation with 50 μMN-(2-ethyl-3-methoxybenzoyl)-N′- (3,5-dimethylbenzoyl)-N′-t- Compositionof Switch butylhydrazine 1.1 GAL4EcR + VP16RXR 267  pGAL4RELuc 1.2GAL4EcR + VP16USP  2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 85 pGAL4RELuc1.4 GAL4RXR + VP16DmEcR 312  pGAL4RELuc 1.5 GAL4USP + VP16CfEcR  2pGAL4RELuc 1.6 GAL4CfEcRVP16  9 pGAL4RELuc 1.7 VP16CfEcR 36 pEcRELuc 1.8DmVgRXR + MmRXR 14 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 27 pIND(SP1)LacZ1.10 CfVgRXR 29 pIND(SP1)LacZ

[0279] TABLE 2 Transactivation of reporter genes through variousswitches in 3T3 cells Mean Fold Activation ThroughN-(2-ethyl-3-methoxybenzoyl)-N′- (3,5-dimethylbenzoyl)- Composition ofSwitch N′-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 1118 pGAL4RELuc 1.2GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 47 pGAL4RELuc 1.4GAL4RXR + VP16DmEcR 269 pGAL4RELuc 1.5 GAL4USP + VP16CfEcR 3 pGAL4RELuc1.6 GAL4CfEcRVP16 7 pGAL4RELuc 1.7 VP16CfEcR 1 pEcRELuc 1.8 DmVgRXR +MmRXR 21 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 19 pIND(SP1)LacZ 1.10 CfVgRXR2 pIND(SP1)LacZ

[0280] TABLE 3 Transactivation of reporter genes through variousswitches in 293 cells Mean Fold Activation ThroughN-(2-ethyl-3-methoxybenzoyl)-N′- (3,5-dimethylbenzoyl)- Composition ofSwitch N′-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 125 pGAL4RELuc 1.2GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 17 pGAL4RELuc 1.4GAL4RXR + VP16DmEcR 3 pGAL4RELuc 1.5 GAL4USP + VP16CfEcR 2 pGAL4RELuc1.6 GAL4CfEcRVP16 3 pGAL4RELuc 1.7 VP16CfEcR 2 pEcRELuc 1.8 DmVgRXR +MmRXR 21 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 12 pIND(SP1)LacZ 1.10 CfVgRXR3 pIND(SP1)LacZ

[0281] TABLE 4 Transactivation of reporter genes through variousswitches in CV1 cells Mean Fold Activation ThroughN-(2-ethyl-3-methoxybenzoyl)-N′- (3,5-dimethylbenzoyl)- Composition ofSwitch N′-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 279 pGAL4RELuc 1.2GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 25 pGAL4RELuc 1.4GAL4RXR + VP16DmEcR 80 pGAL4RELuc 1.5 GAL4USP + VP16CfEcR 3 pGAL4RELuc1.6 GAL4CfEcRVP16 6 pGAL4RELuc 1.7 VP16CfEcR 1 pEcRELuc 1.8 DmVgRXR +MmRXR 12 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR 7 pIND(SP1)LacZ 1.10 CfVgRXR1 pIND(SP1)LacZ

[0282] TABLE 5 Transactivation of reporter geneGAL4CfEcRDEF/VP16MmRXRDEF (switch 1.1) through steroids and non-steroidsin 3T3 cells. Mean Fold Induction at 1.0 μM Ligand Concentration 1.Ponasterone A 1.0 2. Muristerone A 1.0 3.N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5- 116dimethylbenzoyl)-N′-tert-butylhydrazine 4.N′-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)- 601N′-(3,5-dimethylbenzoyl)-N′-tert- butylhydrazine

[0283] TABLE 6 Transactivation of reporter geneGAL4MmRXRDEF/VP16CfEcRCDEF (switch 1.3) through steroids andnon-steroids in 3T3 cells. Mean Fold Induction at 1.0 μM LigandConcentration 1. Ponasterone A 1.0 2. Muristerone A 1.0 3.N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5- 71dimethylbenzoyl)-N′-tert-butylhydrazine 4.N′-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)- 54N′-(3,5-dimethylbenzoyl)-N′-tert- butylhydrazine

[0284] Applicants' results demonstrate that the non-steroidal ecdysoneagonists,N-(2-ethyl-3methoxybenzoyl)N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazineandN′-(3,4(1,2-ethyenedioxy)-2-methylbenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine,were more potent activators of CfEcR as compared to Drosophilamelanogaster EcR (DmEcR). (see Tables 14). Also, in the mammalian celllines tested, MmRXR performed better than CfUSP as a heterodimericpartner for CfEcR. (see Tables 14). Additionally, Applicants' induciblegene expression modulation system performed better when exogenous MmRXRwas used than when the system relied only on endogenous RXR levels (seeTables 14).

[0285] Applicants' results also show that in a CfEcR-based induciblegene expression system, the non-steroidal ecdysone agonist inducedreporter gene expression at a lower concentration (i.e., increasedligand sensitivity) as compared to the steroid ligands, ponasterone Aand muristerone A (see Tables 5 and 6).

[0286] Out of 10 EcR based gene switches tested, the GAL4EcR/VP16RXRswitch (Switch 1.1) performed better than any other switch in all fourcell lines examined and was more sensitive to non-steroids thansteroids. The results also demonstrate that placing the activationdomain (AD) and DNA binding domain (DNABD) on each of the two partnersreduced background when compared to placing both AD and DNABD togetheron one of the two partners. Therefore, a switch format where the AD andDNABD are separated between two partners, works well for EcR-based geneswitch applications.

[0287] In addition, the MmRXR/EcR-based switches performed better thanCfUSP/EcR-based switches, which have a higher background activity thanthe MmRXR/EoR switches in the absence of ligand.

[0288] Finally, the GAL4EcR/VP16RXR switch (Switch 1.1) was moresensitive to nonsteroid ligands than to the steroid ligands (see Tables5 and 6). In particular, steroid ligands initiated transactivation atconcentrations of 50 μM, whereas the non-steroid ligands initiatedtransactivation at less than 1 μM (submicromolar) concentration.

Example 2

[0289] This Example describes Applicants' further analysis of truncatedEcR and RXR polypeptides in the improved EcR-based inducible geneexpression system of the invention. To identify the best combination andlength of two receptors that give a switch with a) maximum induction inthe presence of ligand; b) minimum background in the absence of ligand;c) highly sensitive to ligand concentration; and d) minimum cross-talkamong ligands and receptors, Applicants made and analyzed severaltruncation mutations of the CfEcR and MMRXR receptor polypeptides inNIH3T3 cells.

[0290] Briefly, polynucleotides encoding EcR or RXR receptors weretruncated at the junctions of A/B, C, D, E and F domains and fused toeither a GAL4 DNA binding domain encoding polynucleotide (SEQ ID NO: 41)for CfEcR, or a VP16 activation domain encoding polynucleotide (SEQ IDNO: 45) for MmRXR as described in Example 1. The resulting receptortruncation/fusion polypeptides were assayed in NIH3T3 cells. PlasmidpFRLUC (Stratagene) encoding a luciferase polypeptide was used as areporter gene construct and pTKRL (Promega) encoding a Renillaluciferase polypeptide under the control of the constitutive TK promoterwas used to normalize the transfections as described above. The analysiswas performed in triplicates and mean luciferase counts were determinedas described above.

[0291] Gene Expression Cassettes Encoding Truncated Ecdysone ReceptorPolypeptides

[0292] Gene expression cassettes comprising polynucleotides encodingeither fill length or truncated CfEcR polypeptides fused to a GAL4 DNAbinding domain (SEQ ID NO: 41): GAL4CfEcRA/BCDEF (full lengthCfEcRA/BCDEF; SEQ ID NO: 49), GAL4CfEcRCDEF (CfEcRCDEF; SEQ ID NO: 1),GAIACfEcR1/2CDEF (CfEcR1/2CDEF; SEQ ID NO: 2), GAL4CfEcRDEF (CfEcRDEF;SEQ ID NO: 3), GALACfEcREF (CfEcREF; SEQ ID NO: 4), and GAL4CfEcRDE(CfEcRDE; SEQ ID NO: 5) were wansfected into NIH3T3 cells along withVP16MmRXRDEF (constructed as in Example 1.1; FIG. 11) or VP16MmR EF[constructed as in Example 1.1 except that MmRXRDEF was replaced withMmRXREF (SEQ ID NO: 23); FIG. 12], and pFRLUc and pTKRL plasmid DNAs.The transfected cells were grown in the presence 0, 1, 5 or 25 uM ofN-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazineor PonA for 48 hr. The cells were harvested, lysed and luciferasereporter activity was measured in the cell lysates. Total fly luciferaserelative light units are presented. The number on the top of each bar isthe maximun fold induction for that treatment.

[0293] Applicants' results show that the EF domain of MmRXR issufficient and performs better than DEF domains of this receptor (seeFIGS. 11 and 12). Applicants have also shown that, in general, EcR/RXRreceptor combinations are insensitive to PonA (see FIGS. 11 and 12). Asshown in the FIGS. 11 and 12, the GAL4CfEcRCDEF hybrid polypeptide (SEQID NO: 7) performed better than any other CfEcR hybrid polypeptide.

[0294] Gene Expression Cassettes Encoding Truncated Retinoid X ReceptorPol peptides

[0295] Gene expression cassettes comprising polynucleotides encodingeither full length or truncated MmRXR polypeptides fused to a VP16transactivation domain (SEQ ID NO: 45): VP16MmR A/BCDEF (full lengthMmRXRA/BCDEF; SEQ ID NO: 53), VP16MmRXRCDEF (MmRXRCDEF; SEQ ID NO: 21),VP16MmRDEF (MmRXRDEF; SEQ ID NO: 22), VP16MmRXREF RXREF; SEQ ID NO: 23),VP16MmRXRBam-EF (“MmRXRBam-EF” or “MmRXR-truncatedEF”; SEQ ID NO: 24),and VP16MmRXRAdel (“MmRXRAF2del” or “MmRxR-E”; SEQ ID NO: 25) constructswere transfected into NIH3T3 cells along with GAL4CfEcRCDEF (constructedas in Example 1.1; FIG. 13) or GAL4CfEcRDEF [constructed as in Example1.1 except CfEcRCDEF was replaced with CfEcRDEF (SEQ ID NO: 3); FIG.14], pFRLUc and pTKRL plasmid DNAs as described above. The transfectedcells were grown in the presence 0, 1, 5 and 25 uM ofN-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazineor PonA for 48 hr. The cells were harvested and lysed and reporteractivity was measured in the cell lysate. Total fly luciferase relativelight units are presented. The nuiber on top of each bar is the maximumfold induction in that treatment Of all the truncations of MmRXR tested,Applicants' results show that the MmRXREF receptor was the best partnerfor CfEcR (FIGS. 13 and 14). CfEcRCDEF showed better induction thanCfEcRDEF using MmRXREF. Deleting AF2 (abbreviated “EF-AF2del”) orhelices 1-3 of the E domain (abbreviated “EF-Bamdel”) resulted in an RXRreceptor that reduced gene induction and ligand sensitivity whenpartnered with either CfEcRCDEF (FIG. 13) or CfEcRDEF (FIG. 14) inNIH3T3 cells. In general, the CfEcR/RXR-based switch was much moresensitive to the non-steroidN-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazinethan to the steroid PonA.

Example 3

[0296] This Example describes Applicants' further analysis of geneexpression cassettes encoding truncated EcR or RXR receptor polypeptidesthat affect either ligand binding activity or ligand sensitivity, orboth. Briefly, six different combinations of chimeric receptor pairs,constructed as described in Examples 1 and 2, were further analyzed in asingle experiment in NIH3T3 cells. These six receptor pair combinationsand their corresponding sample numbers are depicted in Table 7. TABLE 7CfEcR + MmRXR Truncation Receptor Combinations in NIH3T3 Cells EcRPolypeptide RXR Polypeptide X-Axis Sample No. Construct ConstructSamples 1 and 2 GAL4CfEcRCDEF VP16RXRA/BCDEF (Full length) Samples 3 and4 GAL4CfEcRCDEF VP16RXRDEF Samples 5 and 6 GAL4CfEcRCDEF VP16RXREFSamples 7 and 8 GAL4CfEcRDEF VP16RXRA/BCDEF (Full length) Samples 9 and10 GAL4CfEcRDEF VP16RXRDEF Samples 11 and 12 GAL4CfEcRDEF VP16RXREF

[0297] The above receptor construct pairs, along with the reporterplasmid pFRLuc were transfected into NIH3T3 cells as described above.The six CfEcR truncation receptor combinations were duplicated into twogroups and treated with either steroid (odd numbers on x-axis of FIG.15) or non-steroid (even numbers on x-axis of FIG. 15). In particular,the cells were grown in media containing 0, 1, 5 or 25 uM PonA (steroid)orN-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine(non-steroid) ligand. The reporter gene activity was measured and totalRLU are shown. Ihe number on top of each bar is the maximum foldinduction for that treatment and is the mean of three replicates.

[0298] As shown in FIG. 15, the CfEcRCDEF/MmRXREF receptor combinationswere the best switch pairs both in terms of total RLU and fold induction(compare columns 1-6 to columns 7-12). This confirms Applicants' earlierfindings as described in Example 2 (FIGS. 11-14). The same geneexpression cassettes encoding the truncated EcR and RXR polypeptideswere also assayed in a human lung carcinoma cell line A549 (ATCC) andsimilar results were observed (data not shown).

1 64 1 1288 DNA Artificial Sequence misc_feature Novel Sequence 1aagggccctg cgccccgtca gcaagaggaa ctgtgtctgg tatgcgggga cagagcctcc 60ggataccact acaatgcgct cacgtgtgaa gggtgtaaag ggttcttcag acggagtgtt 120accaaaaatg cggtttatat ttgtaaattc ggtcacgctt gcgaaatgga catgtacatg 180cgacggaaat gccaggagtg ccgcctgaag aagtgcttag ctgtaggcat gaggcctgag 240tgcgtagtac ccgagactca gtgcgccatg aagcggaaag agaagaaagc acagaaggag 300aaggacaaac tgcctgtcag cacgacgacg gtggacgacc acatgccgcc cattatgcag 360tgtgaacctc cacctcctga agcagcaagg attcacgaag tggtcccaag gtttctctcc 420gacaagctgt tggagacaaa ccggcagaaa aacatccccc agttgacagc caaccagcag 480ttccttatcg ccaggctcat ctggtaccag gacgggtacg agcagccttc tgatgaagat 540ttgaagagga ttacgcagac gtggcagcaa gcggacgatg aaaacgaaga gtctgacact 600cccttccgcc agatcacaga gatgactatc ctcacggtcc aacttatcgt ggagttcgcg 660aagggattgc cagggttcgc caagatctcg cagcctgatc aaattacgct gcttaaggct 720tgctcaagtg aggtaatgat gctccgagtc gcgcgacgat acgatgcggc ctcagacagt 780gttctgttcg cgaacaacca agcgtacact cgcgacaact accgcaaggc tggcatggcc 840tacgtcatcg aggatctact gcacttctgc cggtgcatgt actctatggc gttggacaac 900atccattacg cgctgctcac ggctgtcgtc atcttttctg accggccagg gttggagcag 960ccgcaactgg tggaagaaat ccagcggtac tacctgaata cgctccgcat ctatatcctg 1020aaccagctga gcgggtcggc gcgttcgtcc gtcatatacg gcaagatcct ctcaatcctc 1080tctgagctac gcacgctcgg catgcaaaac tccaacatgt gcatctccct caagctcaag 1140aacagaaagc tgccgccttt cctcgaggag atctgggatg tggcggacat gtcgcacacc 1200caaccgccgc ctatcctcga gtcccccacg aatctctagc ccctgcgcgc acgcatcgcc 1260gatgccgcgt ccggccgcgc tgctctga 1288 2 1110 DNA Artificial Sequencemisc_feature Novel Sequence 2 gcggtttata tttgtaaatt cggtcacgcttgcgaaatgg acatgtacat gcgacggaaa 60 tgccaggagt gccgcctgaa gaagtgcttagctgtaggca tgaggcctga gtgcgtagta 120 cccgagactc agtgcgccat gaagcggaaagagaagaaag cacagaagga gaaggacaaa 180 ctgcctgtca gcacgacgac ggtggacgaccacatgccgc ccattatgca gtgtgaacct 240 ccacctcctg aagcagcaag gattcacgaagtggtcccaa ggtttctctc cgacaagctg 300 ttggagacaa accggcagaa aaacatcccccagttgacag ccaaccagca gttccttatc 360 gccaggctca tctggtacca ggacgggtacgagcagcctt ctgatgaaga tttgaagagg 420 attacgcaga cgtggcagca agcggacgatgaaaacgaag agtctgacac tcccttccgc 480 cagatcacag agatgactat cctcacggtccaacttatcg tggagttcgc gaagggattg 540 ccagggttcg ccaagatctc gcagcctgatcaaattacgc tgcttaaggc ttgctcaagt 600 gaggtaatga tgctccgagt cgcgcgacgatacgatgcgg cctcagacag tgttctgttc 660 gcgaacaacc aagcgtacac tcgcgacaactaccgcaagg ctggcatggc ctacgtcatc 720 gaggatctac tgcacttctg ccggtgcatgtactctatgg cgttggacaa catccattac 780 gcgctgctca cggctgtcgt catcttttctgaccggccag ggttggagca gccgcaactg 840 gtggaagaaa tccagcggta ctacctgaatacgctccgca tctatatcct gaaccagctg 900 agcgggtcgg cgcgttcgtc cgtcatatacggcaagatcc tctcaatcct ctctgagcta 960 cgcacgctcg gcatgcaaaa ctccaacatgtgcatctccc tcaagctcaa gaacagaaag 1020 ctgccgcctt tcctcgagga gatctgggatgtggcggaca tgtcgcacac ccaaccgccg 1080 cctatcctcg agtcccccac gaatctctag1110 3 1054 DNA Artificial Sequence misc_feature Novel Sequence 3cctgagtgcg tagtacccga gactcagtgc gccatgaagc ggaaagagaa gaaagcacag 60aaggagaagg acaaactgcc tgtcagcacg acgacggtgg acgaccacat gccgcccatt 120atgcagtgtg aacctccacc tcctgaagca gcaaggattc acgaagtggt cccaaggttt 180ctctccgaca agctgttgga gacaaaccgg cagaaaaaca tcccccagtt gacagccaac 240cagcagttcc ttatcgccag gctcatctgg taccaggacg ggtacgagca gccttctgat 300gaagatttga agaggattac gcagacgtgg cagcaagcgg acgatgaaaa cgaagagtct 360gacactccct tccgccagat cacagagatg actatcctca cggtccaact tatcgtggag 420ttcgcgaagg gattgccagg gttcgccaag atctcgcagc ctgatcaaat tacgctgctt 480aaggcttgct caagtgaggt aatgatgctc cgagtcgcgc gacgatacga tgcggcctca 540gacagtgttc tgttcgcgaa caaccaagcg tacactcgcg acaactaccg caaggctggc 600atggcctacg tcatcgagga tctactgcac ttctgccggt gcatgtactc tatggcgttg 660gacaacatcc attacgcgct gctcacggct gtcgtcatct tttctgaccg gccagggttg 720gagcagccgc aactggtgga agaaatccag cggtactacc tgaatacgct ccgcatctat 780atcctgaacc agctgagcgg gtcggcgcgt tcgtccgtca tatacggcaa gatcctctca 840atcctctctg agctacgcac gctcggcatg caaaactcca acatgtgcat ctccctcaag 900ctcaagaaca gaaagctgcc gcctttcctc gaggagatct gggatgtggc ggacatgtcg 960cacacccaac cgccgcctat cctcgagtcc cccacgaatc tctagcccct gcgcgcacgc 1020atcgccgatg ccgcgtccgg ccgcgctgct ctga 1054 4 735 DNA Artificial Sequencemisc_feature Novel Sequence 4 taccaggacg ggtacgagca gccttctgatgaagatttga agaggattac gcagacgtgg 60 cagcaagcgg acgatgaaaa cgaagagtctgacactccct tccgccagat cacagagatg 120 actatcctca cggtccaact tatcgtggagttcgcgaagg gattgccagg gttcgccaag 180 atctcgcagc ctgatcaaat tacgctgcttaaggcttgct caagtgaggt aatgatgctc 240 cgagtcgcgc gacgatacga tgcggcctcagacagtgttc tgttcgcgaa caaccaagcg 300 tacactcgcg acaactaccg caaggctggcatggcctacg tcatcgagga tctactgcac 360 ttctgccggt gcatgtactc tatggcgttggacaacatcc attacgcgct gctcacggct 420 gtcgtcatct tttctgaccg gccagggttggagcagccgc aactggtgga agaaatccag 480 cggtactacc tgaatacgct ccgcatctatatcctgaacc agctgagcgg gtcggcgcgt 540 tcgtccgtca tatacggcaa gatcctctcaatcctctctg agctacgcac gctcggcatg 600 caaaactcca acatgtgcat ctccctcaagctcaagaaca gaaagctgcc gcctttcctc 660 gaggagatct gggatgtggc ggacatgtcgcacacccaac cgccgcctat cctcgagtcc 720 cccacgaatc tctag 735 5 960 DNAArtificial Sequence misc_feature Novel Sequence 5 cctgagtgcg tagtacccgagactcagtgc gccatgaagc ggaaagagaa gaaagcacag 60 aaggagaagg acaaactgcctgtcagcacg acgacggtgg acgaccacat gccgcccatt 120 atgcagtgtg aacctccacctcctgaagca gcaaggattc acgaagtggt cccaaggttt 180 ctctccgaca agctgttggagacaaaccgg cagaaaaaca tcccccagtt gacagccaac 240 cagcagttcc ttatcgccaggctcatctgg taccaggacg ggtacgagca gccttctgat 300 gaagatttga agaggattacgcagacgtgg cagcaagcgg acgatgaaaa cgaagagtct 360 gacactccct tccgccagatcacagagatg actatcctca cggtccaact tatcgtggag 420 ttcgcgaagg gattgccagggttcgccaag atctcgcagc ctgatcaaat tacgctgctt 480 aaggcttgct caagtgaggtaatgatgctc cgagtcgcgc gacgatacga tgcggcctca 540 gacagtgttc tgttcgcgaacaaccaagcg tacactcgcg acaactaccg caaggctggc 600 atggcctacg tcatcgaggatctactgcac ttctgccggt gcatgtactc tatggcgttg 660 gacaacatcc attacgcgctgctcacggct gtcgtcatct tttctgaccg gccagggttg 720 gagcagccgc aactggtggaagaaatccag cggtactacc tgaatacgct ccgcatctat 780 atcctgaacc agctgagcgggtcggcgcgt tcgtccgtca tatacggcaa gatcctctca 840 atcctctctg agctacgcacgctcggcatg caaaactcca acatgtgcat ctccctcaag 900 ctcaagaaca gaaagctgccgcctttcctc gaggagatct gggatgtggc ggacatgtcg 960 6 1878 DNA ArtificialSequence misc_feature Novel Sequence 6 ggacctgcgc cacgggtgca agaggagctgtgcctggttt gcggcgacag ggcctccggc 60 taccactaca acgccctcac ctgtgagggctgcaaggggt tctttcgacg cagcgttacg 120 aagagcgccg tctactgctg caagttcgggcgcgcctgcg aaatggacat gtacatgagg 180 cgaaagtgtc aggagtgccg cctgaaaaagtgcctggccg tgggtatgcg gccggaatgc 240 gtcgtcccgg agaaccaatg tgcgatgaagcggcgcgaaa agaaggccca gaaggagaag 300 gacaaaatga ccacttcgcc gagctctcagcatggcggca atggcagctt ggcctctggt 360 ggcggccaag actttgttaa gaaggagattcttgacctta tgacatgcga gccgccccag 420 catgccacta ttccgctact acctgatgaaatattggcca agtgtcaagc gcgcaatata 480 ccttccttaa cgtacaatca gttggccgttatatacaagt taatttggta ccaggatggc 540 tatgagcagc catctgaaga ggatctcaggcgtataatga gtcaacccga tgagaacgag 600 agccaaacgg acgtcagctt tcggcatataaccgagataa ccatactcac ggtccagttg 660 attgttgagt ttgctaaagg tctaccagcgtttacaaaga taccccagga ggaccagatc 720 acgttactaa aggcctgctc gtcggaggtgatgatgctgc gtatggcacg acgctatgac 780 cacagctcgg actcaatatt cttcgcgaataatagatcat atacgcggga ttcttacaaa 840 atggccggaa tggctgataa cattgaagacctgctgcatt tctgccgcca aatgttctcg 900 atgaaggtgg acaacgtcga atacgcgcttctcactgcca ttgtgatctt ctcggaccgg 960 ccgggcctgg agaaggccca actagtcgaagcgatccaga gctactacat cgacacgcta 1020 cgcatttata tactcaaccg ccactgcggcgactcaatga gcctcgtctt ctacgcaaag 1080 ctgctctcga tcctcaccga gctgcgtacgctgggcaacc agaacgccga gatgtgtttc 1140 tcactaaagc tcaaaaaccg caaactgcccaagttcctcg aggagatctg ggacgttcat 1200 gccatcccgc catcggtcca gtcgcaccttcagattaccc aggaggagaa cgagcgtctc 1260 gagcgggctg agcgtatgcg ggcatcggttgggggcgcca ttaccgccgg cattgattgc 1320 gactctgcct ccacttcggc ggcggcagccgcggcccagc atcagcctca gcctcagccc 1380 cagccccaac cctcctccct gacccagaacgattcccagc accagacaca gccgcagcta 1440 caacctcagc taccacctca gctgcaaggtcaactgcaac cccagctcca accacagctt 1500 cagacgcaac tccagccaca gattcaaccacagccacagc tccttcccgt ctccgctccc 1560 gtgcccgcct ccgtaaccgc acctggttccttgtccgcgg tcagtacgag cagcgaatac 1620 atgggcggaa gtgcggccat aggacccatcacgccggcaa ccaccagcag tatcacggct 1680 gccgttaccg ctagctccac cacatcagcggtaccgatgg gcaacggagt tggagtcggt 1740 gttggggtgg gcggcaacgt cagcatgtatgcgaacgccc agacggcgat ggccttgatg 1800 ggtgtagccc tgcattcgca ccaagagcagcttatcgggg gagtggcggt taagtcggag 1860 cactcgacga ctgcatag 1878 7 1752DNA Artificial Sequence misc_feature Novel Sequence 7 gccgtctactgctgcaagtt cgggcgcgcc tgcgaaatgg acatgtacat gaggcgaaag 60 tgtcaggagtgccgcctgaa aaagtgcctg gccgtgggta tgcggccgga atgcgtcgtc 120 ccggagaaccaatgtgcgat gaagcggcgc gaaaagaagg cccagaagga gaaggacaaa 180 atgaccacttcgccgagctc tcagcatggc ggcaatggca gcttggcctc tggtggcggc 240 caagactttgttaagaagga gattcttgac cttatgacat gcgagccgcc ccagcatgcc 300 actattccgctactacctga tgaaatattg gccaagtgtc aagcgcgcaa tataccttcc 360 ttaacgtacaatcagttggc cgttatatac aagttaattt ggtaccagga tggctatgag 420 cagccatctgaagaggatct caggcgtata atgagtcaac ccgatgagaa cgagagccaa 480 acggacgtcagctttcggca tataaccgag ataaccatac tcacggtcca gttgattgtt 540 gagtttgctaaaggtctacc agcgtttaca aagatacccc aggaggacca gatcacgtta 600 ctaaaggcctgctcgtcgga ggtgatgatg ctgcgtatgg cacgacgcta tgaccacagc 660 tcggactcaatattcttcgc gaataataga tcatatacgc gggattctta caaaatggcc 720 ggaatggctgataacattga agacctgctg catttctgcc gccaaatgtt ctcgatgaag 780 gtggacaacgtcgaatacgc gcttctcact gccattgtga tcttctcgga ccggccgggc 840 ctggagaaggcccaactagt cgaagcgatc cagagctact acatcgacac gctacgcatt 900 tatatactcaaccgccactg cggcgactca atgagcctcg tcttctacgc aaagctgctc 960 tcgatcctcaccgagctgcg tacgctgggc aaccagaacg ccgagatgtg tttctcacta 1020 aagctcaaaaaccgcaaact gcccaagttc ctcgaggaga tctgggacgt tcatgccatc 1080 ccgccatcggtccagtcgca ccttcagatt acccaggagg agaacgagcg tctcgagcgg 1140 gctgagcgtatgcgggcatc ggttgggggc gccattaccg ccggcattga ttgcgactct 1200 gcctccacttcggcggcggc agccgcggcc cagcatcagc ctcagcctca gccccagccc 1260 caaccctcctccctgaccca gaacgattcc cagcaccaga cacagccgca gctacaacct 1320 cagctaccacctcagctgca aggtcaactg caaccccagc tccaaccaca gcttcagacg 1380 caactccagccacagattca accacagcca cagctccttc ccgtctccgc tcccgtgccc 1440 gcctccgtaaccgcacctgg ttccttgtcc gcggtcagta cgagcagcga atacatgggc 1500 ggaagtgcggccataggacc catcacgccg gcaaccacca gcagtatcac ggctgccgtt 1560 accgctagctccaccacatc agcggtaccg atgggcaacg gagttggagt cggtgttggg 1620 gtgggcggcaacgtcagcat gtatgcgaac gcccagacgg cgatggcctt gatgggtgta 1680 gccctgcattcgcaccaaga gcagcttatc gggggagtgg cggttaagtc ggagcactcg 1740 acgactgcatag 1752 8 1650 DNA Artificial Sequence misc_feature Novel Sequence 8cggccggaat gcgtcgtccc ggagaaccaa tgtgcgatga agcggcgcga aaagaaggcc 60cagaaggaga aggacaaaat gaccacttcg ccgagctctc agcatggcgg caatggcagc 120ttggcctctg gtggcggcca agactttgtt aagaaggaga ttcttgacct tatgacatgc 180gagccgcccc agcatgccac tattccgcta ctacctgatg aaatattggc caagtgtcaa 240gcgcgcaata taccttcctt aacgtacaat cagttggccg ttatatacaa gttaatttgg 300taccaggatg gctatgagca gccatctgaa gaggatctca ggcgtataat gagtcaaccc 360gatgagaacg agagccaaac ggacgtcagc tttcggcata taaccgagat aaccatactc 420acggtccagt tgattgttga gtttgctaaa ggtctaccag cgtttacaaa gataccccag 480gaggaccaga tcacgttact aaaggcctgc tcgtcggagg tgatgatgct gcgtatggca 540cgacgctatg accacagctc ggactcaata ttcttcgcga ataatagatc atatacgcgg 600gattcttaca aaatggccgg aatggctgat aacattgaag acctgctgca tttctgccgc 660caaatgttct cgatgaaggt ggacaacgtc gaatacgcgc ttctcactgc cattgtgatc 720ttctcggacc ggccgggcct ggagaaggcc caactagtcg aagcgatcca gagctactac 780atcgacacgc tacgcattta tatactcaac cgccactgcg gcgactcaat gagcctcgtc 840ttctacgcaa agctgctctc gatcctcacc gagctgcgta cgctgggcaa ccagaacgcc 900gagatgtgtt tctcactaaa gctcaaaaac cgcaaactgc ccaagttcct cgaggagatc 960tgggacgttc atgccatccc gccatcggtc cagtcgcacc ttcagattac ccaggaggag 1020aacgagcgtc tcgagcgggc tgagcgtatg cgggcatcgg ttgggggcgc cattaccgcc 1080ggcattgatt gcgactctgc ctccacttcg gcggcggcag ccgcggccca gcatcagcct 1140cagcctcagc cccagcccca accctcctcc ctgacccaga acgattccca gcaccagaca 1200cagccgcagc tacaacctca gctaccacct cagctgcaag gtcaactgca accccagctc 1260caaccacagc ttcagacgca actccagcca cagattcaac cacagccaca gctccttccc 1320gtctccgctc ccgtgcccgc ctccgtaacc gcacctggtt ccttgtccgc ggtcagtacg 1380agcagcgaat acatgggcgg aagtgcggcc ataggaccca tcacgccggc aaccaccagc 1440agtatcacgg ctgccgttac cgctagctcc accacatcag cggtaccgat gggcaacgga 1500gttggagtcg gtgttggggt gggcggcaac gtcagcatgt atgcgaacgc ccagacggcg 1560atggccttga tgggtgtagc cctgcattcg caccaagagc agcttatcgg gggagtggcg 1620gttaagtcgg agcactcgac gactgcatag 1650 9 1338 DNA Artificial Sequencemisc_feature Novel Sequence 9 tatgagcagc catctgaaga ggatctcaggcgtataatga gtcaacccga tgagaacgag 60 agccaaacgg acgtcagctt tcggcatataaccgagataa ccatactcac ggtccagttg 120 attgttgagt ttgctaaagg tctaccagcgtttacaaaga taccccagga ggaccagatc 180 acgttactaa aggcctgctc gtcggaggtgatgatgctgc gtatggcacg acgctatgac 240 cacagctcgg actcaatatt cttcgcgaataatagatcat atacgcggga ttcttacaaa 300 atggccggaa tggctgataa cattgaagacctgctgcatt tctgccgcca aatgttctcg 360 atgaaggtgg acaacgtcga atacgcgcttctcactgcca ttgtgatctt ctcggaccgg 420 ccgggcctgg agaaggccca actagtcgaagcgatccaga gctactacat cgacacgcta 480 cgcatttata tactcaaccg ccactgcggcgactcaatga gcctcgtctt ctacgcaaag 540 ctgctctcga tcctcaccga gctgcgtacgctgggcaacc agaacgccga gatgtgtttc 600 tcactaaagc tcaaaaaccg caaactgcccaagttcctcg aggagatctg ggacgttcat 660 gccatcccgc catcggtcca gtcgcaccttcagattaccc aggaggagaa cgagcgtctc 720 gagcgggctg agcgtatgcg ggcatcggttgggggcgcca ttaccgccgg cattgattgc 780 gactctgcct ccacttcggc ggcggcagccgcggcccagc atcagcctca gcctcagccc 840 cagccccaac cctcctccct gacccagaacgattcccagc accagacaca gccgcagcta 900 caacctcagc taccacctca gctgcaaggtcaactgcaac cccagctcca accacagctt 960 cagacgcaac tccagccaca gattcaaccacagccacagc tccttcccgt ctccgctccc 1020 gtgcccgcct ccgtaaccgc acctggttccttgtccgcgg tcagtacgag cagcgaatac 1080 atgggcggaa gtgcggccat aggacccatcacgccggcaa ccaccagcag tatcacggct 1140 gccgttaccg ctagctccac cacatcagcggtaccgatgg gcaacggagt tggagtcggt 1200 gttggggtgg gcggcaacgt cagcatgtatgcgaacgccc agacggcgat ggccttgatg 1260 ggtgtagccc tgcattcgca ccaagagcagcttatcgggg gagtggcggt taagtcggag 1320 cactcgacga ctgcatag 1338 10 969DNA Artificial Sequence misc_feature Novel Sequence 10 cggccggaatgcgtcgtccc ggagaaccaa tgtgcgatga agcggcgcga aaagaaggcc 60 cagaaggagaaggacaaaat gaccacttcg ccgagctctc agcatggcgg caatggcagc 120 ttggcctctggtggcggcca agactttgtt aagaaggaga ttcttgacct tatgacatgc 180 gagccgccccagcatgccac tattccgcta ctacctgatg aaatattggc caagtgtcaa 240 gcgcgcaatataccttcctt aacgtacaat cagttggccg ttatatacaa gttaatttgg 300 taccaggatggctatgagca gccatctgaa gaggatctca ggcgtataat gagtcaaccc 360 gatgagaacgagagccaaac ggacgtcagc tttcggcata taaccgagat aaccatactc 420 acggtccagttgattgttga gtttgctaaa ggtctaccag cgtttacaaa gataccccag 480 gaggaccagatcacgttact aaaggcctgc tcgtcggagg tgatgatgct gcgtatggca 540 cgacgctatgaccacagctc ggactcaata ttcttcgcga ataatagatc atatacgcgg 600 gattcttacaaaatggccgg aatggctgat aacattgaag acctgctgca tttctgccgc 660 caaatgttctcgatgaaggt ggacaacgtc gaatacgcgc ttctcactgc cattgtgatc 720 ttctcggaccggccgggcct ggagaaggcc caactagtcg aagcgatcca gagctactac 780 atcgacacgctacgcattta tatactcaac cgccactgcg gcgactcaat gagcctcgtc 840 ttctacgcaaagctgctctc gatcctcacc gagctgcgta cgctgggcaa ccagaacgcc 900 gagatgtgtttctcactaaa gctcaaaaac cgcaaactgc ccaagttcct cgaggagatc 960 tgggacgtt 96911 412 PRT Artificial Sequence misc_feature Novel Sequence 11 Lys GlyPro Ala Pro Arg Gln Gln Glu Glu Leu Cys Leu Val Cys Gly 1 5 10 15 AspArg Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys 20 25 30 LysGly Phe Phe Arg Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys 35 40 45 LysPhe Gly His Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys 50 55 60 GlnGlu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu 65 70 75 80Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys 85 90 95Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp 100 105110 Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala 115120 125 Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu130 135 140 Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn GlnGln 145 150 155 160 Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly TyrGlu Gln Pro 165 170 175 Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr TrpGln Gln Ala Asp 180 185 190 Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe ArgGln Ile Thr Glu Met 195 200 205 Thr Ile Leu Thr Val Gln Leu Ile Val GluPhe Ala Lys Gly Leu Pro 210 215 220 Gly Phe Ala Lys Ile Ser Gln Pro AspGln Ile Thr Leu Leu Lys Ala 225 230 235 240 Cys Ser Ser Glu Val Met MetLeu Arg Val Ala Arg Arg Tyr Asp Ala 245 250 255 Ala Ser Asp Ser Val LeuPhe Ala Asn Asn Gln Ala Tyr Thr Arg Asp 260 265 270 Asn Tyr Arg Lys AlaGly Met Ala Tyr Val Ile Glu Asp Leu Leu His 275 280 285 Phe Cys Arg CysMet Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala 290 295 300 Leu Leu ThrAla Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln 305 310 315 320 ProGln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg 325 330 335Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile 340 345350 Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met 355360 365 Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu370 375 380 Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser HisThr 385 390 395 400 Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 405410 12 412 PRT Artificial Sequence misc_feature Novel Sequence 12 LysGly Pro Ala Pro Arg Gln Gln Glu Glu Leu Cys Leu Val Cys Gly 1 5 10 15Asp Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys 20 25 30Lys Gly Phe Phe Arg Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys 35 40 45Lys Phe Gly His Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys 50 55 60Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu 65 70 7580 Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys 85 9095 Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp 100105 110 Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala115 120 125 Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys LeuLeu 130 135 140 Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala AsnGln Gln 145 150 155 160 Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp GlyTyr Glu Gln Pro 165 170 175 Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln ThrTrp Gln Gln Ala Asp 180 185 190 Asp Glu Asn Glu Glu Ser Asp Thr Pro PheArg Gln Ile Thr Glu Met 195 200 205 Thr Ile Leu Thr Val Gln Leu Ile ValGlu Phe Ala Lys Gly Leu Pro 210 215 220 Gly Phe Ala Lys Ile Ser Gln ProAsp Gln Ile Thr Leu Leu Lys Ala 225 230 235 240 Cys Ser Ser Glu Val MetMet Leu Arg Val Ala Arg Arg Tyr Asp Ala 245 250 255 Ala Ser Asp Ser ValLeu Phe Ala Asn Asn Gln Ala Tyr Thr Arg Asp 260 265 270 Asn Tyr Arg LysAla Gly Met Ala Tyr Val Ile Glu Asp Leu Leu His 275 280 285 Phe Cys ArgCys Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala 290 295 300 Leu LeuThr Ala Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln 305 310 315 320Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg 325 330335 Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile 340345 350 Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met355 360 365 Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg LysLeu 370 375 380 Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met SerHis Thr 385 390 395 400 Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu405 410 13 334 PRT Artificial Sequence misc_feature Novel Sequence 13Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu 1 5 1015 Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr 20 2530 Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro 35 4045 Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys 50 5560 Leu Leu Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn 65 7075 80 Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu 8590 95 Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln100 105 110 Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln IleThr 115 120 125 Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe AlaLys Gly 130 135 140 Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln IleThr Leu Leu 145 150 155 160 Lys Ala Cys Ser Ser Glu Val Met Met Leu ArgVal Ala Arg Arg Tyr 165 170 175 Asp Ala Ala Ser Asp Ser Val Leu Phe AlaAsn Asn Gln Ala Tyr Thr 180 185 190 Arg Asp Asn Tyr Arg Lys Ala Gly MetAla Tyr Val Ile Glu Asp Leu 195 200 205 Leu His Phe Cys Arg Cys Met TyrSer Met Ala Leu Asp Asn Ile His 210 215 220 Tyr Ala Leu Leu Thr Ala ValVal Ile Phe Ser Asp Arg Pro Gly Leu 225 230 235 240 Glu Gln Pro Gln LeuVal Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr 245 250 255 Leu Arg Ile TyrIle Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser 260 265 270 Val Ile TyrGly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu 275 280 285 Gly MetGln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg 290 295 300 LysLeu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser 305 310 315320 His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 325 330 14244 PRT Artificial Sequence misc_feature Novel Sequence 14 Tyr Gln AspGly Tyr Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile 1 5 10 15 Thr GlnThr Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr 20 25 30 Pro PheArg Gln Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile 35 40 45 Val GluPhe Ala Lys Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro 50 55 60 Asp GlnIle Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu 65 70 75 80 ArgVal Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser Val Leu Phe Ala 85 90 95 AsnAsn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala 100 105 110Tyr Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met 115 120125 Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe 130135 140 Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln145 150 155 160 Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn GlnLeu Ser 165 170 175 Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile LeuSer Ile Leu 180 185 190 Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser AsnMet Cys Ile Ser 195 200 205 Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro PheLeu Glu Glu Ile Trp 210 215 220 Asp Val Ala Asp Met Ser His Thr Gln ProPro Pro Ile Leu Glu Ser 225 230 235 240 Pro Thr Asn Leu 15 320 PRTArtificial Sequence misc_feature Novel Sequence 15 Pro Glu Cys Val ValPro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu 1 5 10 15 Lys Lys Ala GlnLys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr 20 25 30 Val Asp Asp HisMet Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro 35 40 45 Glu Ala Ala ArgIle His Glu Val Val Pro Arg Phe Leu Ser Asp Lys 50 55 60 Leu Leu Glu ThrAsn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn 65 70 75 80 Gln Gln PheLeu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu 85 90 95 Gln Pro SerAsp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln 100 105 110 Ala AspAsp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr 115 120 125 GluMet Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly 130 135 140Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu 145 150155 160 Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg Tyr165 170 175 Asp Ala Ala Ser Asp Ser Val Leu Phe Ala Asn Asn Gln Ala TyrThr 180 185 190 Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile GluAsp Leu 195 200 205 Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu AspAsn Ile His 210 215 220 Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser AspArg Pro Gly Leu 225 230 235 240 Glu Gln Pro Gln Leu Val Glu Glu Ile GlnArg Tyr Tyr Leu Asn Thr 245 250 255 Leu Arg Ile Tyr Ile Leu Asn Gln LeuSer Gly Ser Ala Arg Ser Ser 260 265 270 Val Ile Tyr Gly Lys Ile Leu SerIle Leu Ser Glu Leu Arg Thr Leu 275 280 285 Gly Met Gln Asn Ser Asn MetCys Ile Ser Leu Lys Leu Lys Asn Arg 290 295 300 Lys Leu Pro Pro Phe LeuGlu Glu Ile Trp Asp Val Ala Asp Met Ser 305 310 315 320 16 625 PRTArtificial Sequence misc_feature Novel Sequence 16 Gly Pro Ala Pro ArgVal Gln Glu Glu Leu Cys Leu Val Cys Gly Asp 1 5 10 15 Arg Ala Ser GlyTyr His Tyr Asn Ala Leu Thr Cys Glu Gly Cys Lys 20 25 30 Gly Phe Phe ArgArg Ser Val Thr Lys Ser Ala Val Tyr Cys Cys Lys 35 40 45 Phe Gly Arg AlaCys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln 50 55 60 Glu Cys Arg LeuLys Lys Cys Leu Ala Val Gly Met Arg Pro Glu Cys 65 70 75 80 Val Val ProGlu Asn Gln Cys Ala Met Lys Arg Arg Glu Lys Lys Ala 85 90 95 Gln Lys GluLys Asp Lys Met Thr Thr Ser Pro Ser Ser Gln His Gly 100 105 110 Gly AsnGly Ser Leu Ala Ser Gly Gly Gly Gln Asp Phe Val Lys Lys 115 120 125 GluIle Leu Asp Leu Met Thr Cys Glu Pro Pro Gln His Ala Thr Ile 130 135 140Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln Ala Arg Asn Ile 145 150155 160 Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr Lys Leu Ile Trp165 170 175 Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp Leu Arg ArgIle 180 185 190 Met Ser Gln Pro Asp Glu Asn Glu Ser Gln Thr Asp Val SerPhe Arg 195 200 205 His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln Leu IleVal Glu Phe 210 215 220 Ala Lys Gly Leu Pro Ala Phe Thr Lys Ile Pro GlnGlu Asp Gln Ile 225 230 235 240 Thr Leu Leu Lys Ala Cys Ser Ser Glu ValMet Met Leu Arg Met Ala 245 250 255 Arg Arg Tyr Asp His Ser Ser Asp SerIle Phe Phe Ala Asn Asn Arg 260 265 270 Ser Tyr Thr Arg Asp Ser Tyr LysMet Ala Gly Met Ala Asp Asn Ile 275 280 285 Glu Asp Leu Leu His Phe CysArg Gln Met Phe Ser Met Lys Val Asp 290 295 300 Asn Val Glu Tyr Ala LeuLeu Thr Ala Ile Val Ile Phe Ser Asp Arg 305 310 315 320 Pro Gly Leu GluLys Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr Tyr 325 330 335 Ile Asp ThrLeu Arg Ile Tyr Ile Leu Asn Arg His Cys Gly Asp Ser 340 345 350 Met SerLeu Val Phe Tyr Ala Lys Leu Leu Ser Ile Leu Thr Glu Leu 355 360 365 ArgThr Leu Gly Asn Gln Asn Ala Glu Met Cys Phe Ser Leu Lys Leu 370 375 380Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile Trp Asp Val His 385 390395 400 Ala Ile Pro Pro Ser Val Gln Ser His Leu Gln Ile Thr Gln Glu Glu405 410 415 Asn Glu Arg Leu Glu Arg Ala Glu Arg Met Arg Ala Ser Val GlyGly 420 425 430 Ala Ile Thr Ala Gly Ile Asp Cys Asp Ser Ala Ser Thr SerAla Ala 435 440 445 Ala Ala Ala Ala Gln His Gln Pro Gln Pro Gln Pro GlnPro Gln Pro 450 455 460 Ser Ser Leu Thr Gln Asn Asp Ser Gln His Gln ThrGln Pro Gln Leu 465 470 475 480 Gln Pro Gln Leu Pro Pro Gln Leu Gln GlyGln Leu Gln Pro Gln Leu 485 490 495 Gln Pro Gln Leu Gln Thr Gln Leu GlnPro Gln Ile Gln Pro Gln Pro 500 505 510 Gln Leu Leu Pro Val Ser Ala ProVal Pro Ala Ser Val Thr Ala Pro 515 520 525 Gly Ser Leu Ser Ala Val SerThr Ser Ser Glu Tyr Met Gly Gly Ser 530 535 540 Ala Ala Ile Gly Pro IleThr Pro Ala Thr Thr Ser Ser Ile Thr Ala 545 550 555 560 Ala Val Thr AlaSer Ser Thr Thr Ser Ala Val Pro Met Gly Asn Gly 565 570 575 Val Gly ValGly Val Gly Val Gly Gly Asn Val Ser Met Tyr Ala Asn 580 585 590 Ala GlnThr Ala Met Ala Leu Met Gly Val Ala Leu His Ser His Gln 595 600 605 GluGln Leu Ile Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr 610 615 620Ala 625 17 583 PRT Artificial Sequence misc_feature Novel Sequence 17Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala Cys Glu Met Asp Met Tyr 1 5 1015 Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val 20 2530 Gly Met Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala Met Lys 35 4045 Arg Arg Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met Thr Thr Ser 50 5560 Pro Ser Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly 65 7075 80 Gln Asp Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr Cys Glu Pro 8590 95 Pro Gln His Ala Thr Ile Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys100 105 110 Cys Gln Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln Leu AlaVal 115 120 125 Ile Tyr Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln ProSer Glu 130 135 140 Glu Asp Leu Arg Arg Ile Met Ser Gln Pro Asp Glu AsnGlu Ser Gln 145 150 155 160 Thr Asp Val Ser Phe Arg His Ile Thr Glu IleThr Ile Leu Thr Val 165 170 175 Gln Leu Ile Val Glu Phe Ala Lys Gly LeuPro Ala Phe Thr Lys Ile 180 185 190 Pro Gln Glu Asp Gln Ile Thr Leu LeuLys Ala Cys Ser Ser Glu Val 195 200 205 Met Met Leu Arg Met Ala Arg ArgTyr Asp His Ser Ser Asp Ser Ile 210 215 220 Phe Phe Ala Asn Asn Arg SerTyr Thr Arg Asp Ser Tyr Lys Met Ala 225 230 235 240 Gly Met Ala Asp AsnIle Glu Asp Leu Leu His Phe Cys Arg Gln Met 245 250 255 Phe Ser Met LysVal Asp Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile 260 265 270 Val Ile PheSer Asp Arg Pro Gly Leu Glu Lys Ala Gln Leu Val Glu 275 280 285 Ala IleGln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn 290 295 300 ArgHis Cys Gly Asp Ser Met Ser Leu Val Phe Tyr Ala Lys Leu Leu 305 310 315320 Ser Ile Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn Ala Glu Met 325330 335 Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu340 345 350 Glu Ile Trp Asp Val His Ala Ile Pro Pro Ser Val Gln Ser HisLeu 355 360 365 Gln Ile Thr Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala GluArg Met 370 375 380 Arg Ala Ser Val Gly Gly Ala Ile Thr Ala Gly Ile AspCys Asp Ser 385 390 395 400 Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala GlnHis Gln Pro Gln Pro 405 410 415 Gln Pro Gln Pro Gln Pro Ser Ser Leu ThrGln Asn Asp Ser Gln His 420 425 430 Gln Thr Gln Pro Gln Leu Gln Pro GlnLeu Pro Pro Gln Leu Gln Gly 435 440 445 Gln Leu Gln Pro Gln Leu Gln ProGln Leu Gln Thr Gln Leu Gln Pro 450 455 460 Gln Ile Gln Pro Gln Pro GlnLeu Leu Pro Val Ser Ala Pro Val Pro 465 470 475 480 Ala Ser Val Thr AlaPro Gly Ser Leu Ser Ala Val Ser Thr Ser Ser 485 490 495 Glu Tyr Met GlyGly Ser Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr 500 505 510 Thr Ser SerIle Thr Ala Ala Val Thr Ala Ser Ser Thr Thr Ser Ala 515 520 525 Val ProMet Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly Asn 530 535 540 ValSer Met Tyr Ala Asn Ala Gln Thr Ala Met Ala Leu Met Gly Val 545 550 555560 Ala Leu His Ser His Gln Glu Gln Leu Ile Gly Gly Val Ala Val Lys 565570 575 Ser Glu His Ser Thr Thr Ala 580 18 549 PRT Artificial Sequencemisc_feature Novel Sequence 18 Arg Pro Glu Cys Val Val Pro Glu Asn GlnCys Ala Met Lys Arg Arg 1 5 10 15 Glu Lys Lys Ala Gln Lys Glu Lys AspLys Met Thr Thr Ser Pro Ser 20 25 30 Ser Gln His Gly Gly Asn Gly Ser LeuAla Ser Gly Gly Gly Gln Asp 35 40 45 Phe Val Lys Lys Glu Ile Leu Asp LeuMet Thr Cys Glu Pro Pro Gln 50 55 60 His Ala Thr Ile Pro Leu Leu Pro AspGlu Ile Leu Ala Lys Cys Gln 65 70 75 80 Ala Arg Asn Ile Pro Ser Leu ThrTyr Asn Gln Leu Ala Val Ile Tyr 85 90 95 Lys Leu Ile Trp Tyr Gln Asp GlyTyr Glu Gln Pro Ser Glu Glu Asp 100 105 110 Leu Arg Arg Ile Met Ser GlnPro Asp Glu Asn Glu Ser Gln Thr Asp 115 120 125 Val Ser Phe Arg His IleThr Glu Ile Thr Ile Leu Thr Val Gln Leu 130 135 140 Ile Val Glu Phe AlaLys Gly Leu Pro Ala Phe Thr Lys Ile Pro Gln 145 150 155 160 Glu Asp GlnIle Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met 165 170 175 Leu ArgMet Ala Arg Arg Tyr Asp His Ser Ser Asp Ser Ile Phe Phe 180 185 190 AlaAsn Asn Arg Ser Tyr Thr Arg Asp Ser Tyr Lys Met Ala Gly Met 195 200 205Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys Arg Gln Met Phe Ser 210 215220 Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile 225230 235 240 Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln Leu Val Glu AlaIle 245 250 255 Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr Ile Leu AsnArg His 260 265 270 Cys Gly Asp Ser Met Ser Leu Val Phe Tyr Ala Lys LeuLeu Ser Ile 275 280 285 Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn AlaGlu Met Cys Phe 290 295 300 Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro LysPhe Leu Glu Glu Ile 305 310 315 320 Trp Asp Val His Ala Ile Pro Pro SerVal Gln Ser His Leu Gln Ile 325 330 335 Thr Gln Glu Glu Asn Glu Arg LeuGlu Arg Ala Glu Arg Met Arg Ala 340 345 350 Ser Val Gly Gly Ala Ile ThrAla Gly Ile Asp Cys Asp Ser Ala Ser 355 360 365 Thr Ser Ala Ala Ala AlaAla Ala Gln His Gln Pro Gln Pro Gln Pro 370 375 380 Gln Pro Gln Pro SerSer Leu Thr Gln Asn Asp Ser Gln His Gln Thr 385 390 395 400 Gln Pro GlnLeu Gln Pro Gln Leu Pro Pro Gln Leu Gln Gly Gln Leu 405 410 415 Gln ProGln Leu Gln Pro Gln Leu Gln Thr Gln Leu Gln Pro Gln Ile 420 425 430 GlnPro Gln Pro Gln Leu Leu Pro Val Ser Ala Pro Val Pro Ala Ser 435 440 445Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser Thr Ser Ser Glu Tyr 450 455460 Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr Thr Ser 465470 475 480 Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr Thr Ser Ala ValPro 485 490 495 Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly AsnVal Ser 500 505 510 Met Tyr Ala Asn Ala Gln Thr Ala Met Ala Leu Met GlyVal Ala Leu 515 520 525 His Ser His Gln Glu Gln Leu Ile Gly Gly Val AlaVal Lys Ser Glu 530 535 540 His Ser Thr Thr Ala 545 19 445 PRTArtificial Sequence misc_feature Novel Sequence 19 Tyr Glu Gln Pro SerGlu Glu Asp Leu Arg Arg Ile Met Ser Gln Pro 1 5 10 15 Asp Glu Asn GluSer Gln Thr Asp Val Ser Phe Arg His Ile Thr Glu 20 25 30 Ile Thr Ile LeuThr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu 35 40 45 Pro Ala Phe ThrLys Ile Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys 50 55 60 Ala Cys Ser SerGlu Val Met Met Leu Arg Met Ala Arg Arg Tyr Asp 65 70 75 80 His Ser SerAsp Ser Ile Phe Phe Ala Asn Asn Arg Ser Tyr Thr Arg 85 90 95 Asp Ser TyrLys Met Ala Gly Met Ala Asp Asn Ile Glu Asp Leu Leu 100 105 110 His PheCys Arg Gln Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr 115 120 125 AlaLeu Leu Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu 130 135 140Lys Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu 145 150155 160 Arg Ile Tyr Ile Leu Asn Arg His Cys Gly Asp Ser Met Ser Leu Val165 170 175 Phe Tyr Ala Lys Leu Leu Ser Ile Leu Thr Glu Leu Arg Thr LeuGly 180 185 190 Asn Gln Asn Ala Glu Met Cys Phe Ser Leu Lys Leu Lys AsnArg Lys 195 200 205 Leu Pro Lys Phe Leu Glu Glu Ile Trp Asp Val His AlaIle Pro Pro 210 215 220 Ser Val Gln Ser His Leu Gln Ile Thr Gln Glu GluAsn Glu Arg Leu 225 230 235 240 Glu Arg Ala Glu Arg Met Arg Ala Ser ValGly Gly Ala Ile Thr Ala 245 250 255 Gly Ile Asp Cys Asp Ser Ala Ser ThrSer Ala Ala Ala Ala Ala Ala 260 265 270 Gln His Gln Pro Gln Pro Gln ProGln Pro Gln Pro Ser Ser Leu Thr 275 280 285 Gln Asn Asp Ser Gln His GlnThr Gln Pro Gln Leu Gln Pro Gln Leu 290 295 300 Pro Pro Gln Leu Gln GlyGln Leu Gln Pro Gln Leu Gln Pro Gln Leu 305 310 315 320 Gln Thr Gln LeuGln Pro Gln Ile Gln Pro Gln Pro Gln Leu Leu Pro 325 330 335 Val Ser AlaPro Val Pro Ala Ser Val Thr Ala Pro Gly Ser Leu Ser 340 345 350 Ala ValSer Thr Ser Ser Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly 355 360 365 ProIle Thr Pro Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala 370 375 380Ser Ser Thr Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val Gly 385 390395 400 Val Gly Val Gly Gly Asn Val Ser Met Tyr Ala Asn Ala Gln Thr Ala405 410 415 Met Ala Leu Met Gly Val Ala Leu His Ser His Gln Glu Gln LeuIle 420 425 430 Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr Ala 435440 445 20 323 PRT Artificial Sequence misc_feature Novel Sequence 20Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala Met Lys Arg Arg 1 5 1015 Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met Thr Thr Ser Pro Ser 20 2530 Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly Gln Asp 35 4045 Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr Cys Glu Pro Pro Gln 50 5560 His Ala Thr Ile Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln 65 7075 80 Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr 8590 95 Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp100 105 110 Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn Glu Ser Gln ThrAsp 115 120 125 Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr ValGln Leu 130 135 140 Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe Thr LysIle Pro Gln 145 150 155 160 Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys SerSer Glu Val Met Met 165 170 175 Leu Arg Met Ala Arg Arg Tyr Asp His SerSer Asp Ser Ile Phe Phe 180 185 190 Ala Asn Asn Arg Ser Tyr Thr Arg AspSer Tyr Lys Met Ala Gly Met 195 200 205 Ala Asp Asn Ile Glu Asp Leu LeuHis Phe Cys Arg Gln Met Phe Ser 210 215 220 Met Lys Val Asp Asn Val GluTyr Ala Leu Leu Thr Ala Ile Val Ile 225 230 235 240 Phe Ser Asp Arg ProGly Leu Glu Lys Ala Gln Leu Val Glu Ala Ile 245 250 255 Gln Ser Tyr TyrIle Asp Thr Leu Arg Ile Tyr Ile Leu Asn Arg His 260 265 270 Cys Gly AspSer Met Ser Leu Val Phe Tyr Ala Lys Leu Leu Ser Ile 275 280 285 Leu ThrGlu Leu Arg Thr Leu Gly Asn Gln Asn Ala Glu Met Cys Phe 290 295 300 SerLeu Lys Leu Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile 305 310 315320 Trp Asp Val 21 987 DNA Artificial Sequence misc_feature NovelSequence 21 tgtgctatct gtggggaccg ctcctcaggc aaacactatg gggtatacagttgtgagggc 60 tgcaagggct tcttcaagag gacagtacgc aaagacctga cctacacctgccgagacaac 120 aaggactgcc tgatcgacaa gagacagcgg aaccggtgtc agtactgccgctaccagaag 180 tgcctggcca tgggcatgaa gcgggaagct gtgcaggagg agcggcagcggggcaaggac 240 cggaatgaga acgaggtgga gtccaccagc agtgccaacg aggacatgcctgtagagaag 300 attctggaag ccgagcttgc tgtcgagccc aagactgaga catacgtggaggcaaacatg 360 gggctgaacc ccagctcacc aaatgaccct gttaccaaca tctgtcaagcagcagacaag 420 cagctcttca ctcttgtgga gtgggccaag aggatcccac acttttctgagctgccccta 480 gacgaccagg tcatcctgct acgggcaggc tggaacgagc tgctgatcgcctccttctcc 540 caccgctcca tagctgtgaa agatgggatt ctcctggcca ccggcctgcacgtacaccgg 600 aacagcgctc acagtgctgg ggtgggcgcc atctttgaca gggtgctaacagagctggtg 660 tctaagatgc gtgacatgca gatggacaag acggagctgg gctgcctgcgagccattgtc 720 ctgttcaacc ctgactctaa ggggctctca aaccctgctg aggtggaggcgttgagggag 780 aaggtgtatg cgtcactaga agcgtactgc aaacacaagt accctgagcagccgggcagg 840 tttgccaagc tgctgctccg cctgcctgca ctgcgttcca tcgggctcaagtgcctggag 900 cacctgttct tcttcaagct catcggggac acgcccatcg acaccttcctcatggagatg 960 ctggaggcac cacatcaagc cacctag 987 22 789 DNA ArtificialSequence misc_feature Novel Sequence 22 aagcgggaag ctgtgcagga ggagcggcagcggggcaagg accggaatga gaacgaggtg 60 gagtccacca gcagtgccaa cgaggacatgcctgtagaga agattctgga agccgagctt 120 gctgtcgagc ccaagactga gacatacgtggaggcaaaca tggggctgaa ccccagctca 180 ccaaatgacc ctgttaccaa catctgtcaagcagcagaca agcagctctt cactcttgtg 240 gagtgggcca agaggatccc acacttttctgagctgcccc tagacgacca ggtcatcctg 300 ctacgggcag gctggaacga gctgctgatcgcctccttct cccaccgctc catagctgtg 360 aaagatggga ttctcctggc caccggcctgcacgtacacc ggaacagcgc tcacagtgct 420 ggggtgggcg ccatctttga cagggtgctaacagagctgg tgtctaagat gcgtgacatg 480 cagatggaca agacggagct gggctgcctgcgagccattg tcctgttcaa ccctgactct 540 aaggggctct caaaccctgc tgaggtggaggcgttgaggg agaaggtgta tgcgtcacta 600 gaagcgtact gcaaacacaa gtaccctgagcagccgggca ggtttgccaa gctgctgctc 660 cgcctgcctg cactgcgttc catcgggctcaagtgcctgg agcacctgtt cttcttcaag 720 ctcatcgggg acacgcccat cgacaccttcctcatggaga tgctggaggc accacatcaa 780 gccacctag 789 23 714 DNA ArtificialSequence misc_feature Novel Sequence 23 gccaacgagg acatgcctgt agagaagattctggaagccg agcttgctgt cgagcccaag 60 actgagacat acgtggaggc aaacatggggctgaacccca gctcaccaaa tgaccctgtt 120 accaacatct gtcaagcagc agacaagcagctcttcactc ttgtggagtg ggccaagagg 180 atcccacact tttctgagct gcccctagacgaccaggtca tcctgctacg ggcaggctgg 240 aacgagctgc tgatcgcctc cttctcccaccgctccatag ctgtgaaaga tgggattctc 300 ctggccaccg gcctgcacgt acaccggaacagcgctcaca gtgctggggt gggcgccatc 360 tttgacaggg tgctaacaga gctggtgtctaagatgcgtg acatgcagat ggacaagacg 420 gagctgggct gcctgcgagc cattgtcctgttcaaccctg actctaaggg gctctcaaac 480 cctgctgagg tggaggcgtt gagggagaaggtgtatgcgt cactagaagc gtactgcaaa 540 cacaagtacc ctgagcagcc gggcaggtttgccaagctgc tgctccgcct gcctgcactg 600 cgttccatcg ggctcaagtg cctggagcacctgttcttct tcaagctcat cggggacacg 660 cccatcgaca ccttcctcat ggagatgctggaggcaccac atcaagccac ctag 714 24 536 DNA Artificial Sequencemisc_feature Novel Sequence 24 ggatcccaca cttttctgag ctgcccctagacgaccaggt catcctgcta cgggcaggct 60 ggaacgagct gctgatcgcc tccttctcccaccgctccat agctgtgaaa gatgggattc 120 tcctggccac cggcctgcac gtacaccggaacagcgctca cagtgctggg gtgggcgcca 180 tctttgacag ggtgctaaca gagctggtgtctaagatgcg tgacatgcag atggacaaga 240 cggagctggg ctgcctgcga gccattgtcctgttcaaccc tgactctaag gggctctcaa 300 accctgctga ggtggaggcg ttgagggagaaggtgtatgc gtcactagaa gcgtactgca 360 aacacaagta ccctgagcag ccgggcaggtttgccaagct gctgctccgc ctgcctgcac 420 tgcgttccat cgggctcaag tgcctggagcacctgttctt cttcaagctc atcggggaca 480 cgcccatcga caccttcctc atggagatgctggaggcacc acatcaagcc acctag 536 25 672 DNA Artificial Sequencemisc_feature Novel Sequence 25 gccaacgagg acatgcctgt agagaagattctggaagccg agcttgctgt cgagcccaag 60 actgagacat acgtggaggc aaacatggggctgaacccca gctcaccaaa tgaccctgtt 120 accaacatct gtcaagcagc agacaagcagctcttcactc ttgtggagtg ggccaagagg 180 atcccacact tttctgagct gcccctagacgaccaggtca tcctgctacg ggcaggctgg 240 aacgagctgc tgatcgcctc cttctcccaccgctccatag ctgtgaaaga tgggattctc 300 ctggccaccg gcctgcacgt acaccggaacagcgctcaca gtgctggggt gggcgccatc 360 tttgacaggg tgctaacaga gctggtgtctaagatgcgtg acatgcagat ggacaagacg 420 gagctgggct gcctgcgagc cattgtcctgttcaaccctg actctaaggg gctctcaaac 480 cctgctgagg tggaggcgtt gagggagaaggtgtatgcgt cactagaagc gtactgcaaa 540 cacaagtacc ctgagcagcc gggcaggtttgccaagctgc tgctccgcct gcctgcactg 600 cgttccatcg ggctcaagtg cctggagcacctgttcttct tcaagctcat cggggacacg 660 cccatcgaca cc 672 26 1123 DNAArtificial Sequence misc_feature Novel Sequence 26 tgcgccatct gcggggaccgctcctcaggc aagcactatg gagtgtacag ctgcgagggg 60 tgcaagggct tcttcaagcggacggtgcgc aaggacctga cctacacctg ccgcgacaac 120 aaggactgcc tgattgacaagcggcagcgg aaccggtgcc agtactgccg ctaccagaag 180 tgcctggcca tgggcatgaagcgggaagcc gtgcaggagg agcggcagcg tggcaaggac 240 cggaacgaga atgaggtggagtcgaccagc agcgccaacg aggacatgcc ggtggagagg 300 atcctggagg ctgagctggccgtggagccc aagaccgaga cctacgtgga ggcaaacatg 360 gggctgaacc ccagctcgccgaacgaccct gtcaccaaca tttgccaagc agccgacaaa 420 cagcttttca ccctggtggagtgggccaag cggatcccac acttctcaga gctgcccctg 480 gacgaccagg tcatcctgctgcgggcaggc tggaatgagc tgctcatcgc ctccttctcc 540 caccgctcca tcgccgtgaaggacgggatc ctcctggcca ccgggctgca cgtccaccgg 600 aacagcgccc acagcgcaggggtgggcgcc atctttgaca gggtgctgac ggagcttgtg 660 tccaagatgc gggacatgcagatggacaag acggagctgg gctgcctgcg cgccatcgtc 720 ctctttaacc ctgactccaaggggctctcg aacccggccg aggtggaggc gctgagggag 780 aaggtctatg cgtccttggaggcctactgc aagcacaagt acccagagca gccgggaagg 840 ttcgctaagc tcttgctccgcctgccggct ctgcgctcca tcgggctcaa atgcctggaa 900 catctcttct tcttcaagctcatcggggac acacccattg acaccttcct tatggagatg 960 ctggaggcgc cgcaccaaatgacttaggcc tgcgggccca tcctttgtgc ccacccgttc 1020 tggccaccct gcctggacgccagctgttct tctcagcctg agccctgtcc ctgcccttct 1080 ctgcctggcc tgtttggactttggggcaca gcctgtcact gct 1123 27 925 DNA Artificial Sequencemisc_feature Novel Sequence 27 aagcgggaag ccgtgcagga ggagcggcagcgtggcaagg accggaacga gaatgaggtg 60 gagtcgacca gcagcgccaa cgaggacatgccggtggaga ggatcctgga ggctgagctg 120 gccgtggagc ccaagaccga gacctacgtggaggcaaaca tggggctgaa ccccagctcg 180 ccgaacgacc ctgtcaccaa catttgccaagcagccgaca aacagctttt caccctggtg 240 gagtgggcca agcggatccc acacttctcagagctgcccc tggacgacca ggtcatcctg 300 ctgcgggcag gctggaatga gctgctcatcgcctccttct cccaccgctc catcgccgtg 360 aaggacggga tcctcctggc caccgggctgcacgtccacc ggaacagcgc ccacagcgca 420 ggggtgggcg ccatctttga cagggtgctgacggagcttg tgtccaagat gcgggacatg 480 cagatggaca agacggagct gggctgcctgcgcgccatcg tcctctttaa ccctgactcc 540 aaggggctct cgaacccggc cgaggtggaggcgctgaggg agaaggtcta tgcgtccttg 600 gaggcctact gcaagcacaa gtacccagagcagccgggaa ggttcgctaa gctcttgctc 660 cgcctgccgg ctctgcgctc catcgggctcaaatgcctgg aacatctctt cttcttcaag 720 ctcatcgggg acacacccat tgacaccttccttatggaga tgctggaggc gccgcaccaa 780 atgacttagg cctgcgggcc catcctttgtgcccacccgt tctggccacc ctgcctggac 840 gccagctgtt cttctcagcc tgagccctgtccctgccctt ctctgcctgg cctgtttgga 900 ctttggggca cagcctgtca ctgct 925 28850 DNA Artificial Sequence misc_feature Novel Sequence 28 gccaacgaggacatgccggt ggagaggatc ctggaggctg agctggccgt ggagcccaag 60 accgagacctacgtggaggc aaacatgggg ctgaacccca gctcgccgaa cgaccctgtc 120 accaacatttgccaagcagc cgacaaacag cttttcaccc tggtggagtg ggccaagcgg 180 atcccacacttctcagagct gcccctggac gaccaggtca tcctgctgcg ggcaggctgg 240 aatgagctgctcatcgcctc cttctcccac cgctccatcg ccgtgaagga cgggatcctc 300 ctggccaccgggctgcacgt ccaccggaac agcgcccaca gcgcaggggt gggcgccatc 360 tttgacagggtgctgacgga gcttgtgtcc aagatgcggg acatgcagat ggacaagacg 420 gagctgggctgcctgcgcgc catcgtcctc tttaaccctg actccaaggg gctctcgaac 480 ccggccgaggtggaggcgct gagggagaag gtctatgcgt ccttggaggc ctactgcaag 540 cacaagtacccagagcagcc gggaaggttc gctaagctct tgctccgcct gccggctctg 600 cgctccatcgggctcaaatg cctggaacat ctcttcttct tcaagctcat cggggacaca 660 cccattgacaccttccttat ggagatgctg gaggcgccgc accaaatgac ttaggcctgc 720 gggcccatcctttgtgccca cccgttctgg ccaccctgcc tggacgccag ctgttcttct 780 cagcctgagccctgtccctg cccttctctg cctggcctgt ttggactttg gggcacagcc 840 tgtcactgct850 29 670 DNA Artificial Sequence misc_feature Novel Sequence 29atcccacact tctcagagct gcccctggac gaccaggtca tcctgctgcg ggcaggctgg 60aatgagctgc tcatcgcctc cttctcccac cgctccatcg ccgtgaagga cgggatcctc 120ctggccaccg ggctgcacgt ccaccggaac agcgcccaca gcgcaggggt gggcgccatc 180tttgacaggg tgctgacgga gcttgtgtcc aagatgcggg acatgcagat ggacaagacg 240gagctgggct gcctgcgcgc catcgtcctc tttaaccctg actccaaggg gctctcgaac 300ccggccgagg tggaggcgct gagggagaag gtctatgcgt ccttggaggc ctactgcaag 360cacaagtacc cagagcagcc gggaaggttc gctaagctct tgctccgcct gccggctctg 420cgctccatcg ggctcaaatg cctggaacat ctcttcttct tcaagctcat cggggacaca 480cccattgaca ccttccttat ggagatgctg gaggcgccgc accaaatgac ttaggcctgc 540gggcccatcc tttgtgccca cccgttctgg ccaccctgcc tggacgccag ctgttcttct 600cagcctgagc cctgtccctg cccttctctg cctggcctgt ttggactttg gggcacagcc 660tgtcactgct 670 30 672 DNA Artificial Sequence misc_feature NovelSequence 30 gccaacgagg acatgccggt ggagaggatc ctggaggctg agctggccgtggagcccaag 60 accgagacct acgtggaggc aaacatgggg ctgaacccca gctcgccgaacgaccctgtc 120 accaacattt gccaagcagc cgacaaacag cttttcaccc tggtggagtgggccaagcgg 180 atcccacact tctcagagct gcccctggac gaccaggtca tcctgctgcgggcaggctgg 240 aatgagctgc tcatcgcctc cttctcccac cgctccatcg ccgtgaaggacgggatcctc 300 ctggccaccg ggctgcacgt ccaccggaac agcgcccaca gcgcaggggtgggcgccatc 360 tttgacaggg tgctgacgga gcttgtgtcc aagatgcggg acatgcagatggacaagacg 420 gagctgggct gcctgcgcgc catcgtcctc tttaaccctg actccaaggggctctcgaac 480 ccggccgagg tggaggcgct gagggagaag gtctatgcgt ccttggaggcctactgcaag 540 cacaagtacc cagagcagcc gggaaggttc gctaagctct tgctccgcctgccggctctg 600 cgctccatcg ggctcaaatg cctggaacat ctcttcttct tcaagctcatcggggacaca 660 cccattgaca cc 672 31 328 PRT Artificial Sequencemisc_feature Novel Sequence 31 Cys Ala Ile Cys Gly Asp Arg Ser Ser GlyLys His Tyr Gly Val Tyr 1 5 10 15 Ser Cys Glu Gly Cys Lys Gly Phe PheLys Arg Thr Val Arg Lys Asp 20 25 30 Leu Thr Tyr Thr Cys Arg Asp Asn LysAsp Cys Leu Ile Asp Lys Arg 35 40 45 Gln Arg Asn Arg Cys Gln Tyr Cys ArgTyr Gln Lys Cys Leu Ala Met 50 55 60 Gly Met Lys Arg Glu Ala Val Gln GluGlu Arg Gln Arg Gly Lys Asp 65 70 75 80 Arg Asn Glu Asn Glu Val Glu SerThr Ser Ser Ala Asn Glu Asp Met 85 90 95 Pro Val Glu Lys Ile Leu Glu AlaGlu Leu Ala Val Glu Pro Lys Thr 100 105 110 Glu Thr Tyr Val Glu Ala AsnMet Gly Leu Asn Pro Ser Ser Pro Asn 115 120 125 Asp Pro Val Thr Asn IleCys Gln Ala Ala Asp Lys Gln Leu Phe Thr 130 135 140 Leu Val Glu Trp AlaLys Arg Ile Pro His Phe Ser Glu Leu Pro Leu 145 150 155 160 Asp Asp GlnVal Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile 165 170 175 Ala SerPhe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu 180 185 190 AlaThr Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val 195 200 205Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg 210 215220 Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val 225230 235 240 Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu ValGlu 245 250 255 Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala Tyr CysLys His 260 265 270 Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys Leu LeuLeu Arg Leu 275 280 285 Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu GluHis Leu Phe Phe 290 295 300 Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp ThrPhe Leu Met Glu Met 305 310 315 320 Leu Glu Ala Pro His Gln Ala Thr 32532 262 PRT Artificial Sequence misc_feature Novel Sequence 32 Lys ArgGlu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp Arg Asn 1 5 10 15 GluAsn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val 20 25 30 GluLys Ile Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr 35 40 45 TyrVal Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro 50 55 60 ValThr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val 65 70 75 80Glu Trp Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp 85 90 95Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser 100 105110 Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu Ala Thr 115120 125 Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala130 135 140 Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg AspMet 145 150 155 160 Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala IleVal Leu Phe 165 170 175 Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala GluVal Glu Ala Leu 180 185 190 Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala TyrCys Lys His Lys Tyr 195 200 205 Pro Glu Gln Pro Gly Arg Phe Ala Lys LeuLeu Leu Arg Leu Pro Ala 210 215 220 Leu Arg Ser Ile Gly Leu Lys Cys LeuGlu His Leu Phe Phe Phe Lys 225 230 235 240 Leu Ile Gly Asp Thr Pro IleAsp Thr Phe Leu Met Glu Met Leu Glu 245 250 255 Ala Pro His Gln Ala Thr260 33 237 PRT Artificial Sequence misc_feature Novel Sequence 33 AlaAsn Glu Asp Met Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala 1 5 10 15Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp 65 70 7580 Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 9095 Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100105 110 His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu115 120 125 Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu GlyCys 130 135 140 Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly LeuSer Asn 145 150 155 160 Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val TyrAla Ser Leu Glu 165 170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln ProGly Arg Phe Ala Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg SerIle Gly Leu Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu IleGly Asp Thr Pro Ile Asp Thr 210 215 220 Phe Leu Met Glu Met Leu Glu AlaPro His Gln Ala Thr 225 230 235 34 177 PRT Artificial Sequencemisc_feature Novel Sequence 34 Ile Pro His Phe Ser Glu Leu Pro Leu AspAsp Gln Val Ile Leu Leu 1 5 10 15 Arg Ala Gly Trp Asn Glu Leu Leu IleAla Ser Phe Ser His Arg Ser 20 25 30 Ile Ala Val Lys Asp Gly Ile Leu LeuAla Thr Gly Leu His Val His 35 40 45 Arg Asn Ser Ala His Ser Ala Gly ValGly Ala Ile Phe Asp Arg Val 50 55 60 Leu Thr Glu Leu Val Ser Lys Met ArgAsp Met Gln Met Asp Lys Thr 65 70 75 80 Glu Leu Gly Cys Leu Arg Ala IleVal Leu Phe Asn Pro Asp Ser Lys 85 90 95 Gly Leu Ser Asn Pro Ala Glu ValGlu Ala Leu Arg Glu Lys Val Tyr 100 105 110 Ala Ser Leu Glu Ala Tyr CysLys His Lys Tyr Pro Glu Gln Pro Gly 115 120 125 Arg Phe Ala Lys Leu LeuLeu Arg Leu Pro Ala Leu Arg Ser Ile Gly 130 135 140 Leu Lys Cys Leu GluHis Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr 145 150 155 160 Pro Ile AspThr Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Ala 165 170 175 Thr 35224 PRT Artificial Sequence misc_feature Novel Sequence 35 Ala Asn GluAsp Met Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala 1 5 10 15 Val GluPro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30 Pro SerSer Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45 Lys GlnLeu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60 Ser GluLeu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp 65 70 75 80 AsnGlu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 90 95 AspGly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100 105 110His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120125 Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130135 140 Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn145 150 155 160 Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala SerLeu Glu 165 170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly ArgPhe Ala Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile GlyLeu Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu Ile Gly AspThr Pro Ile Asp Thr 210 215 220 36 328 PRT Artificial Sequencemisc_feature Novel Sequence 36 Cys Ala Ile Cys Gly Asp Arg Ser Ser GlyLys His Tyr Gly Val Tyr 1 5 10 15 Ser Cys Glu Gly Cys Lys Gly Phe PheLys Arg Thr Val Arg Lys Asp 20 25 30 Leu Thr Tyr Thr Cys Arg Asp Asn LysAsp Cys Leu Ile Asp Lys Arg 35 40 45 Gln Arg Asn Arg Cys Gln Tyr Cys ArgTyr Gln Lys Cys Leu Ala Met 50 55 60 Gly Met Lys Arg Glu Ala Val Gln GluGlu Arg Gln Arg Gly Lys Asp 65 70 75 80 Arg Asn Glu Asn Glu Val Glu SerThr Ser Ser Ala Asn Glu Asp Met 85 90 95 Pro Val Glu Arg Ile Leu Glu AlaGlu Leu Ala Val Glu Pro Lys Thr 100 105 110 Glu Thr Tyr Val Glu Ala AsnMet Gly Leu Asn Pro Ser Ser Pro Asn 115 120 125 Asp Pro Val Thr Asn IleCys Gln Ala Ala Asp Lys Gln Leu Phe Thr 130 135 140 Leu Val Glu Trp AlaLys Arg Ile Pro His Phe Ser Glu Leu Pro Leu 145 150 155 160 Asp Asp GlnVal Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile 165 170 175 Ala SerPhe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu 180 185 190 AlaThr Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val 195 200 205Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg 210 215220 Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val 225230 235 240 Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu ValGlu 245 250 255 Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala Tyr CysLys His 260 265 270 Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala Lys Leu LeuLeu Arg Leu 275 280 285 Pro Ala Leu Arg Ser Ile Gly Leu Lys Cys Leu GluHis Leu Phe Phe 290 295 300 Phe Lys Leu Ile Gly Asp Thr Pro Ile Asp ThrPhe Leu Met Glu Met 305 310 315 320 Leu Glu Ala Pro His Gln Met Thr 32537 262 PRT Artificial Sequence misc_feature Novel Sequence 37 Lys ArgGlu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp Arg Asn 1 5 10 15 GluAsn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val 20 25 30 GluArg Ile Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr 35 40 45 TyrVal Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro 50 55 60 ValThr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val 65 70 75 80Glu Trp Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp 85 90 95Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser 100 105110 Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu Ala Thr 115120 125 Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala130 135 140 Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg AspMet 145 150 155 160 Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala IleVal Leu Phe 165 170 175 Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala GluVal Glu Ala Leu 180 185 190 Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala TyrCys Lys His Lys Tyr 195 200 205 Pro Glu Gln Pro Gly Arg Phe Ala Lys LeuLeu Leu Arg Leu Pro Ala 210 215 220 Leu Arg Ser Ile Gly Leu Lys Cys LeuGlu His Leu Phe Phe Phe Lys 225 230 235 240 Leu Ile Gly Asp Thr Pro IleAsp Thr Phe Leu Met Glu Met Leu Glu 245 250 255 Ala Pro His Gln Met Thr260 38 237 PRT Artificial Sequence misc_feature Novel Sequence 38 AlaAsn Glu Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala 1 5 10 15Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp 65 70 7580 Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 9095 Asp Gly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100105 110 His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu115 120 125 Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu GlyCys 130 135 140 Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly LeuSer Asn 145 150 155 160 Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val TyrAla Ser Leu Glu 165 170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln ProGly Arg Phe Ala Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg SerIle Gly Leu Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu IleGly Asp Thr Pro Ile Asp Thr 210 215 220 Phe Leu Met Glu Met Leu Glu AlaPro His Gln Met Thr 225 230 235 39 177 PRT Artificial Sequencemisc_feature Novel Sequence 39 Ile Pro His Phe Ser Glu Leu Pro Leu AspAsp Gln Val Ile Leu Leu 1 5 10 15 Arg Ala Gly Trp Asn Glu Leu Leu IleAla Ser Phe Ser His Arg Ser 20 25 30 Ile Ala Val Lys Asp Gly Ile Leu LeuAla Thr Gly Leu His Val His 35 40 45 Arg Asn Ser Ala His Ser Ala Gly ValGly Ala Ile Phe Asp Arg Val 50 55 60 Leu Thr Glu Leu Val Ser Lys Met ArgAsp Met Gln Met Asp Lys Thr 65 70 75 80 Glu Leu Gly Cys Leu Arg Ala IleVal Leu Phe Asn Pro Asp Ser Lys 85 90 95 Gly Leu Ser Asn Pro Ala Glu ValGlu Ala Leu Arg Glu Lys Val Tyr 100 105 110 Ala Ser Leu Glu Ala Tyr CysLys His Lys Tyr Pro Glu Gln Pro Gly 115 120 125 Arg Phe Ala Lys Leu LeuLeu Arg Leu Pro Ala Leu Arg Ser Ile Gly 130 135 140 Leu Lys Cys Leu GluHis Leu Phe Phe Phe Lys Leu Ile Gly Asp Thr 145 150 155 160 Pro Ile AspThr Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Met 165 170 175 Thr 40224 PRT Artificial Sequence misc_feature Novel Sequence 40 Ala Asn GluAsp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala 1 5 10 15 Val GluPro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30 Pro SerSer Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35 40 45 Lys GlnLeu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe 50 55 60 Ser GluLeu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp 65 70 75 80 AsnGlu Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Ala Val Lys 85 90 95 AspGly Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala 100 105 110His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120125 Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130135 140 Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn145 150 155 160 Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala SerLeu Glu 165 170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly ArgPhe Ala Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile GlyLeu Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu Ile Gly AspThr Pro Ile Asp Thr 210 215 220 41 441 DNA Artificial Sequencemisc_feature Novel Sequence 41 atgaagctac tgtcttctat cgaacaagcatgcgatattt gccgacttaa aaagctcaag 60 tgctccaaag aaaaaccgaa gtgcgccaagtgtctgaaga acaactggga gtgtcgctac 120 tctcccaaaa ccaaaaggtc tccgctgactagggcacatc tgacagaagt ggaatcaagg 180 ctagaaagac tggaacagct atttctactgatttttcctc gagaagacct tgacatgatt 240 ttgaaaatgg attctttaca ggatataaaagcattgttaa caggattatt tgtacaagat 300 aatgtgaata aagatgccgt cacagatagattggcttcag tggagactga tatgcctcta 360 acattgagac agcatagaat aagtgcgacatcatcatcgg aagagagtag taacaaaggt 420 caaagacagt tgactgtatc g 441 42 147PRT Artificial Sequence misc_feature Novel Sequence 42 Met Lys Leu LeuSer Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu 1 5 10 15 Lys Lys LeuLys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 Lys Asn AsnTrp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45 Leu Thr ArgAla His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60 Glu Gln LeuPhe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile 65 70 75 80 Leu LysMet Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95 Phe ValGln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110 SerVal Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115 120 125Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu 130 135140 Thr Val Ser 145 43 606 DNA Artificial Sequence misc_feature NovelSequence 43 atgaaagcgt taacggccag gcaacaagag gtgtttgatc tcatccgtgatcacatcagc 60 cagacaggta tgccgccgac gcgtgcggaa atcgcgcagc gtttggggttccgttcccca 120 aacgcggctg aagaacatct gaaggcgctg gcacgcaaag gcgttattgaaattgtttcc 180 ggcgcatcac gcgggattcg tctgttgcag gaagaggaag aagggttgccgctggtaggt 240 cgtgtggctg ccggtgaacc acttctggcg caacagcata ttgaaggtcattatcaggtc 300 gatccttcct tattcaagcc gaatgctgat ttcctgctgc gcgtcagcgggatgtcgatg 360 aaagatatcg gcattatgga tggtgacttg ctggcagtgc ataaaactcaggatgtacgt 420 aacggtcagg tcgttgtcgc acgtattgat gacgaagtta ccgttaagcgcctgaaaaaa 480 cagggcaata aagtcgaact gttgccagaa aatagcgagt ttaaaccaattgtcgtagat 540 cttcgtcagc agagcttcac cattgaaggg ctggcggttg gggttattcgcaacggcgac 600 tggctg 606 44 202 PRT Artificial Sequence misc_featureNovel Sequence 44 Met Lys Ala Leu Thr Ala Arg Gln Gln Glu Val Phe AspLeu Ile Arg 1 5 10 15 Asp His Ile Ser Gln Thr Gly Met Pro Pro Thr ArgAla Glu Ile Ala 20 25 30 Gln Arg Leu Gly Phe Arg Ser Pro Asn Ala Ala GluGlu His Leu Lys 35 40 45 Ala Leu Ala Arg Lys Gly Val Ile Glu Ile Val SerGly Ala Ser Arg 50 55 60 Gly Ile Arg Leu Leu Gln Glu Glu Glu Glu Gly LeuPro Leu Val Gly 65 70 75 80 Arg Val Ala Ala Gly Glu Pro Leu Leu Ala GlnGln His Ile Glu Gly 85 90 95 His Tyr Gln Val Asp Pro Ser Leu Phe Lys ProAsn Ala Asp Phe Leu 100 105 110 Leu Arg Val Ser Gly Met Ser Met Lys AspIle Gly Ile Met Asp Gly 115 120 125 Asp Leu Leu Ala Val His Lys Thr GlnAsp Val Arg Asn Gly Gln Val 130 135 140 Val Val Ala Arg Ile Asp Asp GluVal Thr Val Lys Arg Leu Lys Lys 145 150 155 160 Gln Gly Asn Lys Val GluLeu Leu Pro Glu Asn Ser Glu Phe Lys Pro 165 170 175 Ile Val Val Asp LeuArg Gln Gln Ser Phe Thr Ile Glu Gly Leu Ala 180 185 190 Val Gly Val IleArg Asn Gly Asp Trp Leu 195 200 45 271 DNA Artificial Sequencemisc_feature Novel Sequence 45 atgggcccta aaaagaagcg taaagtcgcccccccgaccg atgtcagcct gggggacgag 60 ctccacttag acggcgagga cgtggcgatggcgcatgccg acgcgctaga cgatttcgat 120 ctggacatgt tgggggacgg ggattccccggggccgggat ttacccccca cgactccgcc 180 ccctacggcg ctctggatat ggccgacttcgagtttgagc agatgtttac cgatgccctt 240 ggaattgacg agtacggtgg ggaattcccg g271 46 90 PRT Artificial Sequence misc_feature Novel Sequence 46 Met GlyPro Lys Lys Lys Arg Lys Val Ala Pro Pro Thr Asp Val Ser 1 5 10 15 LeuGly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala His 20 25 30 AlaAsp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Asp Gly Asp 35 40 45 SerPro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly Ala 50 55 60 LeuAsp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr Asp Ala Leu 65 70 75 80Gly Ile Asp Glu Tyr Gly Gly Glu Phe Pro 85 90 47 19 DNA ArtificialSequence misc_feature Novel Sequence 47 ggagtactgt cctccgagc 19 48 666DNA Artificial Sequence misc_feature Novel Sequence 48 ggatccccagcttggaattc gacaggttat cagcaacaac acagtcatat ccattctcaa 60 ttagctctaccacagtgtgt gaaccaatgt atccagcacc acctgtaacc aaaacaattt 120 tagaagtactttcactttgt aactgagctg tcatttatat tgaattttca aaaattctta 180 ctttttttttggatggacgc aaagaagttt aataatcata ttacatggca ttaccaccat 240 atacatatccatatacatat ccatatctaa tcttacctcg actgctgtat ataaaaccag 300 tggttatatgtacagtactg ctgtatataa aaccagtggt tatatgtaca gtacgtcgac 360 tgctgtatataaaaccagtg gttatatgta cagtactgct gtatataaaa ccagtggtta 420 tatgtacagtacgtcgaggg atgataatgc gattagtttt ttagccttat ttctggggta 480 attaatcagcgaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca 540 taaccactttaactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta 600 ataaaagtatcaacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa 660 actata 666 491542 DNA Artificial Sequence misc_feature Novel Sequence 49 ctggacctgaaacacgaagt ggcttaccga ggggtgctcc caggccaggt gaaggccgaa 60 ccgggggtccacaacggcca ggtcaacggc cacgtgaggg actggatggc aggcggcgct 120 ggtgccaattcgccgtctcc gggagcggtg gctcaacccc agcctaacaa tgggtattcg 180 tcgccactctcctcgggaag ctacgggccc tacagtccaa atgggaaaat aggccgtgag 240 gaactgtcgccagcttcaag tataaatggg tgcagtacag atggcgaggc acgacgtcag 300 aagaagggccctgcgccccg tcagcaagag gaactgtgtc tggtatgcgg ggacagagcc 360 tccggataccactacaatgc gctcacgtgt gaagggtgta aagggttctt cagacggagt 420 gttaccaaaaatgcggttta tatttgtaaa ttcggtcacg cttgcgaaat ggacatgtac 480 atgcgacggaaatgccagga gtgccgcctg aagaagtgct tagctgtagg catgaggcct 540 gagtgcgtagtacccgagac tcagtgcgcc atgaagcgga aagagaagaa agcacagaag 600 gagaaggacaaactgcctgt cagcacgacg acggtggacg accacatgcc gcccattatg 660 cagtgtgaacctccacctcc tgaagcagca aggattcacg aagtggtccc aaggtttctc 720 tccgacaagctgttggagac aaaccggcag aaaaacatcc cccagttgac agccaaccag 780 cagttccttatcgccaggct catctggtac caggacgggt acgagcagcc ttctgatgaa 840 gatttgaagaggattacgca gacgtggcag caagcggacg atgaaaacga agagtctgac 900 actcccttccgccagatcac agagatgact atcctcacgg tccaacttat cgtggagttc 960 gcgaagggattgccagggtt cgccaagatc tcgcagcctg atcaaattac gctgcttaag 1020 gcttgctcaagtgaggtaat gatgctccga gtcgcgcgac gatacgatgc ggcctcagac 1080 agtgttctgttcgcgaacaa ccaagcgtac actcgcgaca actaccgcaa ggctggcatg 1140 gcctacgtcatcgaggatct actgcacttc tgccggtgca tgtactctat ggcgttggac 1200 aacatccattacgcgctgct cacggctgtc gtcatctttt ctgaccggcc agggttggag 1260 cagccgcaactggtggaaga aatccagcgg tactacctga atacgctccg catctatatc 1320 ctgaaccagctgagcgggtc ggcgcgttcg tccgtcatat acggcaagat cctctcaatc 1380 ctctctgagctacgcacgct cggcatgcaa aactccaaca tgtgcatctc cctcaagctc 1440 aagaacagaaagctgccgcc tttcctcgag gagatctggg atgtggcgga catgtcgcac 1500 acccaaccgccgcctatcct cgagtccccc acgaatctct ag 1542 50 513 PRT Artificial Sequencemisc_feature Novel Sequence 50 Leu Asp Leu Lys His Glu Val Ala Tyr ArgGly Val Leu Pro Gly Gln 1 5 10 15 Val Lys Ala Glu Pro Gly Val His AsnGly Gln Val Asn Gly His Val 20 25 30 Arg Asp Trp Met Ala Gly Gly Ala GlyAla Asn Ser Pro Ser Pro Gly 35 40 45 Ala Val Ala Gln Pro Gln Pro Asn AsnGly Tyr Ser Ser Pro Leu Ser 50 55 60 Ser Gly Ser Tyr Gly Pro Tyr Ser ProAsn Gly Lys Ile Gly Arg Glu 65 70 75 80 Glu Leu Ser Pro Ala Ser Ser IleAsn Gly Cys Ser Thr Asp Gly Glu 85 90 95 Ala Arg Arg Gln Lys Lys Gly ProAla Pro Arg Gln Gln Glu Glu Leu 100 105 110 Cys Leu Val Cys Gly Asp ArgAla Ser Gly Tyr His Tyr Asn Ala Leu 115 120 125 Thr Cys Glu Gly Cys LysGly Phe Phe Arg Arg Ser Val Thr Lys Asn 130 135 140 Ala Val Tyr Ile CysLys Phe Gly His Ala Cys Glu Met Asp Met Tyr 145 150 155 160 Met Arg ArgLys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val 165 170 175 Gly MetArg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys 180 185 190 ArgLys Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser 195 200 205Thr Thr Thr Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro 210 215220 Pro Pro Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu 225230 235 240 Ser Asp Lys Leu Leu Glu Thr Asn Arg Gln Lys Asn Ile Pro GlnLeu 245 250 255 Thr Ala Asn Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp TyrGln Asp 260 265 270 Gly Tyr Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg IleThr Gln Thr 275 280 285 Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser AspThr Pro Phe Arg 290 295 300 Gln Ile Thr Glu Met Thr Ile Leu Thr Val GlnLeu Ile Val Glu Phe 305 310 315 320 Ala Lys Gly Leu Pro Gly Phe Ala LysIle Ser Gln Pro Asp Gln Ile 325 330 335 Thr Leu Leu Lys Ala Cys Ser SerGlu Val Met Met Leu Arg Val Ala 340 345 350 Arg Arg Tyr Asp Ala Ala SerAsp Ser Val Leu Phe Ala Asn Asn Gln 355 360 365 Ala Tyr Thr Arg Asp AsnTyr Arg Lys Ala Gly Met Ala Tyr Val Ile 370 375 380 Glu Asp Leu Leu HisPhe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp 385 390 395 400 Asn Ile HisTyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg 405 410 415 Pro GlyLeu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr 420 425 430 LeuAsn Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala 435 440 445Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu 450 455460 Arg Thr Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu 465470 475 480 Lys Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp ValAla 485 490 495 Asp Met Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser ProThr Asn 500 505 510 Leu 51 4375 DNA Artificial Sequence misc_featureNovel Sequence 51 tgtaattttg atgggcgccg tgatgcaccg tgtgccatat tgccatccagtcgaatagaa 60 aaaaaaaaaa aaaaaaaaat atcagttgtt ttgtccctcg ctcgctttcgagtgtattcg 120 gaatattaga cgtcataatt cacgagtgtc ttttaaattt atatagcgattagcggggcc 180 gtttgttgga cgtgcgcttg cgtttagtgg agtgcaggga tagtgaggcgagtatggtag 240 ttcgtggtca tgtcaagtgt ggcgaagaaa gacaagccga cgatgtcggtgacggcgctg 300 atcaactggg cgcggccggc gccgccaggc ccgccgcagc cgcagtcagcgtcgcctgcg 360 ccggcagcca tgctgcagca gctcccgacg cagtcaatgc agtcgttaaaccacatccca 420 actgtcgatt gctcgctcga tatgcagtgg cttaatttag aacctggattcatgtcgcct 480 atgtcacctc ctgagatgaa accagacacc gccatgcttg atgggctacgagacgacgcc 540 acttcgccgc ctaacttcaa gaactacccg cctaatcacc ccctgagtggctccaaacac 600 ctatgctcta tatgcggcga cagggcgtct gggaagcact atggggtgtacagttgcgaa 660 ggatgcaagg gtttcttcaa gcggaccgtc cggaaggacc tgtcgtacgcttgccgggag 720 gagcggaact gcatcataga caagcgacaa aggaaccgat gccagtactgccgctatcaa 780 aagtgtttgg cttgcggtat gaagcgagag gcggtgcaag aggagcgccagaggaatgct 840 cgcggcgcgg aggatgcgca cccgagtagc tcggtgcagg taagcgatgagctgtcaatc 900 gagcgcctaa cggagatgga gtctttggtg gcagatccca gcgaggagttccagttcctc 960 cgcgtggggc ctgacagcaa cgtgcctcca cgttaccgcg cgcccgtctcctccctctgc 1020 caaataggca acaagcaaat agcggcgttg gtggtatggg cgcgcgacatccctcatttc 1080 gggcagctgg agctggacga tcaagtggta ctcatcaagg cctcctggaatgagctgcta 1140 ctcttcgcca tcgcctggcg ctctatggag tatttggaag atgagagggagaacggggac 1200 ggaacgcgga gcaccactca gccacaactg atgtgtctca tgcctggcatgacgttgcac 1260 cgcaactcgg cgcagcaggc gggcgtgggc gccatcttcg accgcgtgctgtccgagctc 1320 agtctgaaga tgcgcacctt gcgcatggac caggccgagt acgtcgcgctcaaagccatc 1380 gtgctgctca accctgatgt gaaaggactg aagaatcggc aagaagttgacgttttgcga 1440 gaaaaaatgt tctcttgcct ggacgactac tgccggcggt cgcgaagcaacgaggaaggc 1500 cggtttgcgt ccttgctgct gcggctgcca gctctccgct ccatctcgctcaagagcttc 1560 gaacacctct acttcttcca cctcgtggcc gaaggctcca tcagcggatacatacgagag 1620 gcgctccgaa accacgcgcc tccgatcgac gtcaatgcca tgatgtaaagtgcgatacac 1680 gccctgccga tgtgagaaga actatggcta atagaagcga aactgaatacatctagggtg 1740 ggacttaact tgggactatc attaaagtat cacgcaaatt atgcgtagtcagaaagtcgc 1800 gtcgatcaaa cttttttata aacgaattga gtttctaacg actgcaacacagcggagttt 1860 tgcttctgat agtttttatt ctaatggtta agatgcttta cacgggcattattgacattc 1920 aagtgtaagt ggaagttgac aaccttgaca tttatatcac gtttgtaattggttaaataa 1980 attaattaat cacaagtaag actaacatca acgtcacgat actaacgccatttagtgata 2040 tttttcatgt caagaaactc attgttttga taaaatattt ttctaattactccagtgaac 2100 tcatccaaat gtgacccagt ttcccgcaga gttgcccgtg taaaatcatctttagggaca 2160 tatcccccgc tatctcatga aattccaagg atcagtaggg gccaattcccccgatgtgtt 2220 gggaggcaga attttcgata atctacgact attgttagcc tacgaattagttgaattttt 2280 tgaaattatt tttattaagt cgccactttc caaacacatc agcagggtatatgtgcaatt 2340 ttgtaacgat aactctattc atttctgata tttatcgaaa ttttatcttacataacatgc 2400 tggctggtcc aggtgtttgg tagttacata tgtatctacg gtttgttttaaattatagct 2460 tttttattgt aatctgtata aaattgagtt atcttacttc acactacgatcgagtaaacc 2520 catcgtcagc tacgaaaaac taatcgtata aggcgtaaga gtaaataactaattgacaac 2580 cagcaacgag gaccacctca gtcctcgtgc ttacattgtg ccgtagcttaatatgatgga 2640 agctgtcgtc gttacgacat tagataaagt gcatgaatac caaaaatgtaccatcccgta 2700 ctgatctctc atgctctcgc tgcgtgggac ccgtgtcgag tgtcgtaaggactgactaat 2760 attttagact aggcgtctat gcttcagtaa ttccttatac atattataagtcatccaaat 2820 aacgagtaag gcggcatgtt gagatcagca ttccgagagt caaagagcccctaacgtgac 2880 tgagaagtag agacaataca ctgattttct gagatgaacg caaccgagattgacactaaa 2940 aatctattta tggatttcaa aatggcgatg cttgattgtc tgcggcgtggatagactgaa 3000 atgggtttgc ttaacactgg atattgtttt tattagttaa tagtcttacattgcaagttg 3060 gtaattcggt gctaatatcg accggtttgt taactatcta acggttcccagtgtcaggca 3120 cacatctttc ccaagcagac aacgcaagag tgtacaaaat gtacatgttacaaaataagg 3180 aacattcgtc ggataagtgt aacagttgat aggtaaagaa aatggggccgcctctttatt 3240 attacgtagc cgtaaaatta ttaacgtatt tagtttagat gttcagctaattaggataat 3300 tctatttgtc gagtacctag atgtccatag tgaattaata taataattagactgttacgc 3360 gtaggtaatt ataaagttta ccaaatctct cttcaaagca aaaactttgtacacttccgt 3420 actgagacgt cgtagcttat tctgattcac gaaatatttg gatcacattgttacaaggcg 3480 accgtcacgt agtatatgat tatttacaaa tgacacgtat gtatcaatgctataagtgtt 3540 ttcgttacat atgtcggtgc tttaacgtgc atttcgatgt gcagattaaaaatagcaaga 3600 aatcttgaaa ttgttttaga aaatatttga tttccttatt gaaagttatttttaaatgta 3660 aatatttcgt aatcataata attatgtatt gtgtagttat ttcacctttacggttgggat 3720 attatttaat ggtggcctac gaaagtgatt ataaccatcc gcgtcctcaaaaaggccagt 3780 ttatttttgt acctcataca tactaattac gtaagtaata tcaggcgaatggttgactaa 3840 caactaacca gtattaaaaa ttaaaagact tcgtcctaat aaaatgtaatatctatgtat 3900 aaaaatgaaa aatctggcgt ataataggta aaattaaact agattgttaatgaatgtgat 3960 gtctcataaa cgtttagttt ttaatgagaa acatgtttag tcgcctactataagacgaga 4020 cggcaagctc accgagttaa ctcgtaaaca ggaatgttga aaaagatgacacaatttata 4080 tttggtattg aaattatgac taaccatgcg ctctatcgtt tgttatggatgcatagtatt 4140 gctgttgaaa ataatggaat taggtaatta ctgcattaat gttgaaaacttgatattatt 4200 ctatggttgg gtatgaattc tatgttggaa gtgttgcagc ggttgtaaagatgatttata 4260 atgatgttca ctaaatatct gactaaatgt aagttatttt tttttgtatagacatagctt 4320 taagatgaag gtgattaaac tttatcctta tcacaataaa aaaaaaaaaaaaaaa 4375 52 472 PRT Artificial Sequence misc_feature Novel Sequence 52Met Ser Ser Val Ala Lys Lys Asp Lys Pro Thr Met Ser Val Thr Ala 1 5 1015 Leu Ile Asn Trp Ala Arg Pro Ala Pro Pro Gly Pro Pro Gln Pro Gln 20 2530 Ser Ala Ser Pro Ala Pro Ala Ala Met Leu Gln Gln Leu Pro Thr Gln 35 4045 Ser Met Gln Ser Leu Asn His Ile Pro Thr Val Asp Cys Ser Leu Asp 50 5560 Met Gln Trp Leu Asn Leu Glu Pro Gly Phe Met Ser Pro Met Ser Pro 65 7075 80 Pro Glu Met Lys Pro Asp Thr Ala Met Leu Asp Gly Leu Arg Asp Asp 8590 95 Ala Thr Ser Pro Pro Asn Phe Lys Asn Tyr Pro Pro Asn His Pro Leu100 105 110 Ser Gly Ser Lys His Leu Cys Ser Ile Cys Gly Asp Arg Ala SerGly 115 120 125 Lys His Tyr Gly Val Tyr Ser Cys Glu Gly Cys Lys Gly PhePhe Lys 130 135 140 Arg Thr Val Arg Lys Asp Leu Ser Tyr Ala Cys Arg GluGlu Arg Asn 145 150 155 160 Cys Ile Ile Asp Lys Arg Gln Arg Asn Arg CysGln Tyr Cys Arg Tyr 165 170 175 Gln Lys Cys Leu Ala Cys Gly Met Lys ArgGlu Ala Val Gln Glu Glu 180 185 190 Arg Gln Arg Asn Ala Arg Gly Ala GluAsp Ala His Pro Ser Ser Ser 195 200 205 Val Gln Val Ser Asp Glu Leu SerIle Glu Arg Leu Thr Glu Met Glu 210 215 220 Ser Leu Val Ala Asp Pro SerGlu Glu Phe Gln Phe Leu Arg Val Gly 225 230 235 240 Pro Asp Ser Asn ValPro Pro Arg Tyr Arg Ala Pro Val Ser Ser Leu 245 250 255 Cys Gln Ile GlyAsn Lys Gln Ile Ala Ala Leu Val Val Trp Ala Arg 260 265 270 Asp Ile ProHis Phe Gly Gln Leu Glu Leu Asp Asp Gln Val Val Leu 275 280 285 Ile LysAla Ser Trp Asn Glu Leu Leu Leu Phe Ala Ile Ala Trp Arg 290 295 300 SerMet Glu Tyr Leu Glu Asp Glu Arg Glu Asn Gly Asp Gly Thr Arg 305 310 315320 Ser Thr Thr Gln Pro Gln Leu Met Cys Leu Met Pro Gly Met Thr Leu 325330 335 His Arg Asn Ser Ala Gln Gln Ala Gly Val Gly Ala Ile Phe Asp Arg340 345 350 Val Leu Ser Glu Leu Ser Leu Lys Met Arg Thr Leu Arg Met AspGln 355 360 365 Ala Glu Tyr Val Ala Leu Lys Ala Ile Val Leu Leu Asn ProAsp Val 370 375 380 Lys Gly Leu Lys Asn Arg Gln Glu Val Asp Val Leu ArgGlu Lys Met 385 390 395 400 Phe Ser Cys Leu Asp Asp Tyr Cys Arg Arg SerArg Ser Asn Glu Glu 405 410 415 Gly Arg Phe Ala Ser Leu Leu Leu Arg LeuPro Ala Leu Arg Ser Ile 420 425 430 Ser Leu Lys Ser Phe Glu His Leu TyrPhe Phe His Leu Val Ala Glu 435 440 445 Gly Ser Ile Ser Gly Tyr Ile ArgGlu Ala Leu Arg Asn His Ala Pro 450 455 460 Pro Ile Asp Val Asn Ala MetMet 465 470 53 1404 DNA Artificial Sequence misc_feature Novel Sequence53 atggacacca aacatttcct gccgctcgac ttctctaccc aggtgaactc ttcgtccctc 60aactctccaa cgggtcgagg ctccatggct gtcccctcgc tgcacccctc cttgggtccg 120ggaatcggct ctccactggg ctcgcctggg cagctgcact ctcctatcag caccctgagc 180tcccccatca atggcatggg tccgcccttc tctgtcatca gctcccccat gggcccgcac 240tccatgtcgg tacccaccac acccacattg ggcttcggga ctggtagccc ccagctcaat 300tcacccatga accctgtgag cagcactgag gatatcaagc cgccactagg cctcaatggc 360gtcctcaagg ttcctgccca tccctcagga aatatggcct ccttcaccaa gcacatctgt 420gctatctgtg gggaccgctc ctcaggcaaa cactatgggg tatacagttg tgagggctgc 480aagggcttct tcaagaggac agtacgcaaa gacctgacct acacctgccg agacaacaag 540gactgcctga tcgacaagag acagcggaac cggtgtcagt actgccgcta ccagaagtgc 600ctggccatgg gcatgaagcg ggaagctgtg caggaggagc ggcagcgggg caaggaccgg 660aatgagaacg aggtggagtc caccagcagt gccaacgagg acatgcctgt agagaagatt 720ctggaagccg agcttgctgt cgagcccaag actgagacat acgtggaggc aaacatgggg 780ctgaacccca gctcaccaaa tgaccctgtt accaacatct gtcaagcagc agacaagcag 840ctcttcactc ttgtggagtg ggccaagagg atcccacact tttctgagct gcccctagac 900gaccaggtca tcctgctacg ggcaggctgg aacgagctgc tgatcgcctc cttctcccac 960cgctccatag ctgtgaaaga tgggattctc ctggccaccg gcctgcacgt acaccggaac 1020agcgctcaca gtgctggggt gggcgccatc tttgacaggg tgctaacaga gctggtgtct 1080aagatgcgtg acatgcagat ggacaagacg gagctgggct gcctgcgagc cattgtcctg 1140ttcaaccctg actctaaggg gctctcaaac cctgctgagg tggaggcgtt gagggagaag 1200gtgtatgcgt cactagaagc gtactgcaaa cacaagtacc ctgagcagcc gggcaggttt 1260gccaagctgc tgctccgcct gcctgcactg cgttccatcg ggctcaagtg cctggagcac 1320ctgttcttct tcaagctcat cggggacacg cccatcgaca ccttcctcat ggagatgctg 1380gaggcaccac atcaagccac ctag 1404 54 467 PRT Artificial Sequencemisc_feature Novel Sequence 54 Met Asp Thr Lys His Phe Leu Pro Leu AspPhe Ser Thr Gln Val Asn 1 5 10 15 Ser Ser Ser Leu Asn Ser Pro Thr GlyArg Gly Ser Met Ala Val Pro 20 25 30 Ser Leu His Pro Ser Leu Gly Pro GlyIle Gly Ser Pro Leu Gly Ser 35 40 45 Pro Gly Gln Leu His Ser Pro Ile SerThr Leu Ser Ser Pro Ile Asn 50 55 60 Gly Met Gly Pro Pro Phe Ser Val IleSer Ser Pro Met Gly Pro His 65 70 75 80 Ser Met Ser Val Pro Thr Thr ProThr Leu Gly Phe Gly Thr Gly Ser 85 90 95 Pro Gln Leu Asn Ser Pro Met AsnPro Val Ser Ser Thr Glu Asp Ile 100 105 110 Lys Pro Pro Leu Gly Leu AsnGly Val Leu Lys Val Pro Ala His Pro 115 120 125 Ser Gly Asn Met Ala SerPhe Thr Lys His Ile Cys Ala Ile Cys Gly 130 135 140 Asp Arg Ser Ser GlyLys His Tyr Gly Val Tyr Ser Cys Glu Gly Cys 145 150 155 160 Lys Gly PhePhe Lys Arg Thr Val Arg Lys Asp Leu Thr Tyr Thr Cys 165 170 175 Arg AspAsn Lys Asp Cys Leu Ile Asp Lys Arg Gln Arg Asn Arg Cys 180 185 190 GlnTyr Cys Arg Tyr Gln Lys Cys Leu Ala Met Gly Met Lys Arg Glu 195 200 205Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp Arg Asn Glu Asn Glu 210 215220 Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val Glu Lys Ile 225230 235 240 Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr Tyr ValGlu 245 250 255 Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro ValThr Asn 260 265 270 Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu ValGlu Trp Ala 275 280 285 Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu AspAsp Gln Val Ile 290 295 300 Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu IleAla Ser Phe Ser His 305 310 315 320 Arg Ser Ile Ala Val Lys Asp Gly IleLeu Leu Ala Thr Gly Leu His 325 330 335 Val His Arg Asn Ser Ala His SerAla Gly Val Gly Ala Ile Phe Asp 340 345 350 Arg Val Leu Thr Glu Leu ValSer Lys Met Arg Asp Met Gln Met Asp 355 360 365 Lys Thr Glu Leu Gly CysLeu Arg Ala Ile Val Leu Phe Asn Pro Asp 370 375 380 Ser Lys Gly Leu SerAsn Pro Ala Glu Val Glu Ala Leu Arg Glu Lys 385 390 395 400 Val Tyr AlaSer Leu Glu Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln 405 410 415 Pro GlyArg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser 420 425 430 IleGly Leu Lys Cys Leu Glu His Leu Phe Phe Phe Lys Leu Ile Gly 435 440 445Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ala Pro His 450 455460 Gln Ala Thr 465 55 309 DNA Artificial Sequence misc_feature NovelSequence 55 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatgcatctcaatt 60 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagtatgcaaagca 120 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatcccgcccctaa 180 ctccgcccag ttccgcccat tctccgcccc atggctgact aattttttttatttatgcag 240 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggcttttttggag 300 gcctaggct 309 56 24 DNA Artificial Sequence misc_featureNovel Sequence 56 tatataatgg atccccgggt accg 24 57 1653 DNA ArtificialSequence misc_feature Novel Sequence 57 atggaagacg ccaaaaacat aaagaaaggcccggcgccat tctatcctct agaggatgga 60 accgctggag agcaactgca taaggctatgaagagatacg ccctggttcc tggaacaatt 120 gcttttacag atgcacatat cgaggtgaacatcacgtacg cggaatactt cgaaatgtcc 180 gttcggttgg cagaagctat gaaacgatatgggctgaata caaatcacag aatcgtcgta 240 tgcagtgaaa actctcttca attctttatgccggtgttgg gcgcgttatt tatcggagtt 300 gcagttgcgc ccgcgaacga catttataatgaacgtgaat tgctcaacag tatgaacatt 360 tcgcagccta ccgtagtgtt tgtttccaaaaaggggttgc aaaaaatttt gaacgtgcaa 420 aaaaaattac caataatcca gaaaattattatcatggatt ctaaaacgga ttaccaggga 480 tttcagtcga tgtacacgtt cgtcacatctcatctacctc ccggttttaa tgaatacgat 540 tttgtaccag agtcctttga tcgtgacaaaacaattgcac tgataatgaa ttcctctgga 600 tctactgggt tacctaaggg tgtggcccttccgcatagaa ctgcctgcgt cagattctcg 660 catgccagag atcctatttt tggcaatcaaatcattccgg atactgcgat tttaagtgtt 720 gttccattcc atcacggttt tggaatgtttactacactcg gatatttgat atgtggattt 780 cgagtcgtct taatgtatag atttgaagaagagctgtttt tacgatccct tcaggattac 840 aaaattcaaa gtgcgttgct agtaccaaccctattttcat tcttcgccaa aagcactctg 900 attgacaaat acgatttatc taatttacacgaaattgctt ctgggggcgc acctctttcg 960 aaagaagtcg gggaagcggt tgcaaaacgcttccatcttc cagggatacg acaaggatat 1020 gggctcactg agactacatc agctattctgattacacccg agggggatga taaaccgggc 1080 gcggtcggta aagttgttcc attttttgaagcgaaggttg tggatctgga taccgggaaa 1140 acgctgggcg ttaatcagag aggcgaattatgtgtcagag gacctatgat tatgtccggt 1200 tatgtaaaca atccggaagc gaccaacgccttgattgaca aggatggatg gctacattct 1260 ggagacatag cttactggga cgaagacgaacacttcttca tagttgaccg cttgaagtct 1320 ttaattaaat acaaaggata tcaggtggcccccgctgaat tggaatcgat attgttacaa 1380 caccccaaca tcttcgacgc gggcgtggcaggtcttcccg acgatgacgc cggtgaactt 1440 cccgccgccg ttgttgtttt ggagcacggaaagacgatga cggaaaaaga gatcgtggat 1500 tacgtcgcca gtcaagtaac aaccgcgaaaaagttgcgcg gaggagttgt gtttgtggac 1560 gaagtaccga aaggtcttac cggaaaactcgacgcaagaa aaatcagaga gatcctcata 1620 aaggccaaga agggcggaaa gtccaaattgtaa 1653 58 867 DNA Artificial Sequence misc_feature Novel Sequence 58aagcgagagg cggtgcaaga ggagcgccag aggaatgctc gcggcgcgga ggatgcgcac 60ccgagtagct cggtgcaggt aagcgatgag ctgtcaatcg agcgcctaac ggagatggag 120tctttggtgg cagatcccag cgaggagttc cagttcctcc gcgtggggcc tgacagcaac 180gtgcctccac gttaccgcgc gcccgtctcc tccctctgcc aaataggcaa caagcaaata 240gcggcgttgg tggtatgggc gcgcgacatc cctcatttcg ggcagctgga gctggacgat 300caagtggtac tcatcaaggc ctcctggaat gagctgctac tcttcgccat cgcctggcgc 360tctatggagt atttggaaga tgagagggag aacggggacg gaacgcggag caccactcag 420ccacaactga tgtgtctcat gcctggcatg acgttgcacc gcaactcggc gcagcaggcg 480ggcgtgggcg ccatcttcga ccgcgtgctg tccgagctca gtctgaagat gcgcaccttg 540cgcatggacc aggccgagta cgtcgcgctc aaagccatcg tgctgctcaa ccctgatgtg 600aaaggactga agaatcggca agaagttgac gttttgcgag aaaaaatgtt ctcttgcctg 660gacgactact gccggcggtc gcgaagcaac gaggaaggcc ggtttgcgtc cttgctgctg 720cggctgccag ctctccgctc catctcgctc aagagcttcg aacacctcta cttcttccac 780ctcgtggccg aaggctccat cagcggatac atacgagagg cgctccgaaa ccacgcgcct 840ccgatcgacg tcaatgccat gatgtaa 867 59 225 DNA Artificial Sequencemisc_feature Novel Sequence 59 tcgacattgg acaagtgcat tgaacccttgtctctcgaga gacaaggggg ttcaatgcac 60 ttgtccaatg tcgagagaca agggggttcaatgcacttgt ccaatgtcga gagacaaggg 120 ggttcaatgc acttgtccaa tgtcgagagacaagggggtt caatgcactt gtccaatgtc 180 gagagacaag ggggttcaat gcacttgtccaatgtcgact ctaga 225 60 619 DNA Artificial Sequence misc_feature NovelSequence 60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacccccgcccatt 60 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttccattgacgtca 120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgtatcatatgcc 180 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcattatgcccagta 240 catgacctta tgggactttc ctacttggca gtacatctac gtattagtcatcgctattac 300 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttgactcacgggg 360 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcaccaaaatcaacg 420 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcggtaggcgtgt 480 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcgcctggagacg 540 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcctccgcggccg 600 ggaacggtgc attggaacg 619 61 262 DNA Artificial Sequencemisc_feature Novel Sequence 61 atgtagtctt atgcaatact cttgtagtcttgcaacatgg taacgatgag ttagcaacat 60 gccttacaag gagagaaaaa gcaccgtgcatgccgatagg tggaagtaag gtggtacgat 120 cgtgccttat taggaaggca acagacgggtctgacatgga ttggacgaac cactgaattc 180 cgcattgcag agatattgta tttaagtgcctagctcgata caataaacgc catttgacca 240 ttcaccacat tggagtgcac ct 262 621247 DNA Artificial Sequence misc_feature Novel Sequence 62 tctatttcctcaggccgtga ggaactgtcg ccagcttcaa gtataaatgg gtgcagtaca 60 gatggcgaggcacgacgtca gaagaagggc cctgcgcccc gtcagcaaga ggaactgtgt 120 ctggtatgcggggacagagc ctccggatac cactacaatg cgctcacgtg tgaagggtgt 180 aaagggttcttcagacggag tgttaccaaa aatgcggttt atatttgtaa attcggtcac 240 gcttgcgaaatggacatgta catgcgacgg aaatgccagg agtgccgcct gaagaagtgc 300 ttagctgtaggcatgaggcc tgagtgcgta gtacccgaga ctcagtgcgc catgaagcgg 360 aaagagaagaaagcacagaa ggagaaggac aaactgcctg tcagcacgac gacggtggac 420 gaccacatgccgcccattat gcagtgtgaa cctccacctc ctgaagcagc aaggattcac 480 gaagtggtcccaaggtttct ctccgacaag ctgttggaga caaaccggca gaaaaacatc 540 ccccagttgacagccaacca gcagttcctt atcgccaggc tcatctggta ccaggacggg 600 tacgagcagccttctgatga agatttgaag aggattacgc agacgtggca gcaagcggac 660 gatgaaaacgaagagtctga cactcccttc cgccagatca cagagatgac tatcctcacg 720 gtccaacttatcgtggagtt cgcgaaggga ttgccagggt tcgccaagat ctcgcagcct 780 gatcaaattacgctgcttaa ggcttgctca agtgaggtaa tgatgctccg agtcgcgcga 840 cgatacgatgcggcctcaga cagtgttctg ttcgcgaaca accaagcgta cactcgcgac 900 aactaccgcaaggctggcat ggcctacgtc atcgaggatc tactgcactt ctgccggtgc 960 atgtactctatggcgttgga caacatccat tacgcgctgc tcacggctgt cgtcatcttt 1020 tctgaccggccagggttgga gcagccgcaa ctggtggaag aaatccagcg gtactacctg 1080 aatacgctccgcatctatat cctgaaccag ctgagcgggt cggcgcgttc gtccgtcata 1140 tacggcaagatcctctcaat cctctctgag ctacgcacgc tcggcatgca aaactccaac 1200 atgtgcatctccctcaagct caagaacaga aagctgccgc ctttcct 1247 63 440 PRT ArtificialSequence misc_feature Novel Sequence 63 Ser Ile Ser Ser Gly Arg Glu GluLeu Ser Pro Ala Ser Ser Ile Asn 1 5 10 15 Gly Cys Ser Thr Asp Gly GluAla Arg Arg Gln Lys Lys Gly Pro Ala 20 25 30 Pro Arg Gln Gln Glu Glu LeuCys Leu Val Cys Gly Asp Arg Ala Ser 35 40 45 Gly Tyr His Tyr Asn Ala LeuThr Cys Glu Gly Cys Lys Gly Phe Phe 50 55 60 Arg Arg Ser Val Thr Lys AsnAla Val Tyr Ile Cys Lys Phe Gly His 65 70 75 80 Ala Cys Glu Met Asp MetTyr Met Arg Arg Lys Cys Gln Glu Cys Arg 85 90 95 Leu Lys Lys Cys Leu AlaVal Gly Met Arg Pro Glu Cys Val Val Pro 100 105 110 Glu Thr Gln Cys AlaMet Lys Arg Lys Glu Lys Lys Ala Gln Lys Glu 115 120 125 Lys Asp Lys LeuPro Val Ser Thr Thr Thr Val Asp Asp His Met Pro 130 135 140 Pro Ile MetGln Cys Glu Pro Pro Pro Pro Glu Ala Ala Arg Ile His 145 150 155 160 GluVal Val Pro Arg Phe Leu Ser Asp Lys Leu Leu Glu Thr Asn Arg 165 170 175Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala 180 185190 Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp 195200 205 Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu210 215 220 Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met Thr Ile LeuThr 225 230 235 240 Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro GlyPhe Ala Lys 245 250 255 Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys AlaCys Ser Ser Glu 260 265 270 Val Met Met Leu Arg Val Ala Arg Arg Tyr AspAla Ala Ser Asp Ser 275 280 285 Val Leu Phe Ala Asn Asn Gln Ala Tyr ThrArg Asp Asn Tyr Arg Lys 290 295 300 Ala Gly Met Ala Tyr Val Ile Glu AspLeu Leu His Phe Cys Arg Cys 305 310 315 320 Met Tyr Ser Met Ala Leu AspAsn Ile His Tyr Ala Leu Leu Thr Ala 325 330 335 Val Val Ile Phe Ser AspArg Pro Gly Leu Glu Gln Pro Gln Leu Val 340 345 350 Glu Glu Ile Gln ArgTyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu 355 360 365 Asn Gln Leu SerGly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile 370 375 380 Leu Ser IleLeu Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn 385 390 395 400 MetCys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu 405 410 415Glu Glu Ile Trp Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro 420 425430 Ile Leu Glu Ser Pro Thr Asn Leu 435 440 64 943 DNA ArtificialSequence misc_feature Novel Sequence 64 atgacttcga aagtttatga tccagaacaaaggaaacgga tgataactgg tccgcagtgg 60 tgggccagat gtaaacaaat gaatgttcttgattcattta ttaattatta tgattcagaa 120 aaacatgcag aaaatgctgt tatttttttacatggtaacg cggcctcttc ttatttatgg 180 cgacatgttg tgccacatat tgagccagtagcgcggtgta ttataccaga ccttattggt 240 atgggcaaat caggcaaatc tggtaatggttcttataggt tacttgatca ttacaaatat 300 cttactgcat ggtttgaact tcttaatttaccaaagaaga tcatttttgt cggccatgat 360 tggggtgctt gtttggcatt tcattatagctatgagcatc aagataagat caaagcaata 420 gttcacgctg aaagtgtagt agatgtgattgaatcatggg atgaatggcc tgatattgaa 480 gaagatattg cgttgatcaa atctgaagaaggagaaaaaa tggttttgga gaataacttc 540 ttcgtggaaa ccatgttgcc atcaaaaatcatgagaaagt tagaaccaga agaatttgca 600 gcatatcttg aaccattcaa agagaaaggtgaagttcgtc gtccaacatt atcatggcct 660 cgtgaaatcc cgttagtaaa aggtggtaaacctgacgttg tacaaattgt taggaattat 720 aatgcttatc tacgtgcaag tgatgatttaccaaaaatgt ttattgaatc ggacccagga 780 ttcttttcca atgctattgt tgaaggtgccaagaagtttc ctaatactga atttgtcaaa 840 gtaaaaggtc ttcatttttc gcaagaagatgcacctgatg aaatgggaaa atatatcaaa 900 tcgttcgttg agcgagttct caaaaatgaacaataattct aga 943

We claim:
 1. A gene expression modulation system comprising: a) a firstgene expression cassette that is capable of being expressed in a hostcell comprising a polynucleotide sequence that encodes a firstpolypeptide comprising: i) a DNA-binding domain that recognizes aresponse element associated with a gene whose expression is to bemodulated; i) a ligand binding domain comprising a ligand binding domainfrom a nuclear receptor; b) a second gene expression cassette that iscapable of being expressed in the host cell comprising a polynucleotidesequence that encodes a second polypeptide comprising: i) atransactivation domain; and ii) a ligand binding domain comprising aligand binding domain from a nuclear receptor other than ultraspiracle(USP); wherein the transactivation domain is from a nuclear receptorother than an ecdysone receptor, a retinoid X receptor, or anultraspiracle receptor; and wherein the ligand binding domains from thefirst polypeptide and the second polypeptide are different and dimerize.2. The gene expression modulation system according to claim 1, furthercomprising a third gene expression cassette comprising: i) a responseelement to which the DNA-binding domain of the first polypeptide binds;ii) a promoter that is activated by the transactivation domain of thesecond polypeptide; and iii) the gene whose expression is to bemodulated.
 3. The gene expression modulation system according to claim1, wherein the ligand binding domain of the first polypeptide is anecdysone receptor polypeptide.
 4. The gene expression modulation systemaccording to claim 1, wherein the ligand binding domain of the secondpolypeptide is a retinoid X receptor polypeptide.
 5. A gene expressionmodulation system comprising: a) a first gene expression cassette thatis capable of being expressed in a host cell comprising a polynucleotidesequence that encodes a first polypeptide comprising: i) a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and ii) a ligand binding domaincomprising a ligand binding domain from an ecdysone receptor; and b) asecond gene expression cassette that is capable of being expressed inthe host cell comprising a polynucleotide sequence that encodes a secondpolypeptide comprising: i) a transactivation domain; and ii) a ligandbinding domain comprising a ligand binding domain from a retinoid Xreceptor; wherein the ligand binding domains from the first polypeptideand the second polypeptide are different and dimerize.
 6. The geneexpression modulation system according to claim 5, further comprising athird gene expression cassette comprising: i) a response element towhich the DNA-binding domain of the fust polypeptide binds; ii) apromoter that is activated by the transactivation domain of the secondpolypeptide; and iii) the gene whose expression is to be modulated. 7.The gene expression modulation system according to claim 5, wherein theligand binding domain of the first polypeptide is encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,and SEQ D NO:
 10. 8. The gene expression modulation system according toclaim 5, wherein the ligand binding domain of the first polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQID NO:20.
 9. The gene expression modulation system according to claim 5,wherein the ligand binding domain of the second polypeptide is encodedby a polynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO:29, and SEQ ID NO:
 30. 10. The gene expression modulation systemaccording to claim 5, wherein the ligand binding domain of the secondpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33,, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO:
 40. 11. A gene expression modulation systemcomprising: a) a first gene expression cassette that is capable of beingexpressed in a host cell comprising a polynucleotide sequence thatencodes a first polypeptide comprising: i) a DNA-binding domain thatrecognizes a response element associated with a gene whose expression isto be modulated; and ii) a ligand binding domain comprising a ligandbinding domain from a retinoid X receptor; and b) a second geneexpression cassette that is capable of being expressed in the host cellcomprising a polynucleotide sequence that encodes a second polypeptidecomprising: i) a transactivation domain; and ii) a ligand binding domaincomprising a ligand binding domain from an ecdysone receptor; whereinthe ligand binding domains from the first polypeptide and the secondpolypeptide are different and dimerize.
 12. The gene expressionmodulation system according to claim 11, further comprising a third geneexpression cassette comprising: i) a response element to which theDNA-binding domain of the first polypeptide binds; ii) a promoter thatis activated by the transactivation domain of the second polypeptide;and iii) the gene whose expression is to be modulated.
 13. The geneexpression modulation system according to claim 11, wherein the ligandbinding domain of the first polypeptide is encoded by a polynucleotidecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQID NO:
 30. 14. The gene expression modulation system according to claim11, wherein the ligand binding domain of the first polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.15. The gene expression modulation system according to claim 11, whereinthe ligand binding domain of the second polypeptide is encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,and SEQ ID NO:
 10. 16. The gene expression modulation system accordingto claim 11, wherein the ligand binding domain of the second polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQID NO:
 20. 17. A gene expression cassette comprising a polynucleotideencoding a hybrid polypeptide comprising a DNA-binding domain and anecdysone receptor ligand binding domain, wherein the DNA binding domainis from a nuclear receptor other than an ecdysone receptor.
 18. The geneexpression cassette according to claim 18, wherein the DNA-bindingdomain is a GAL4 DNA-binding domain or a LexA DNA-binding domain.
 19. Agene expression cassette comprising a polynucleotide encoding a hybridpolypeptide comprising a DNA-binding domain and a retinoid X receptorligand binding domain, wherein the DNA binding domain is from a nuclearreceptor other than a retinoid X receptor.
 20. The gene expressioncassette according to claim 19, wherein the DNA-binding domain is a GALADNA-binding domain or a LexA DNA-binding domain.
 21. A gene expressioncassette comprising a polynucleotide encoding a hybrid polypeptidecomprising a transactivation domain and an ecdysone receptor ligandbinding domain, wherein the transactivation domain is from a nuclearreceptor other than an ecdysone receptor.
 22. The gene expressioncassette according to claim 21, wherein the transactivation domain is aVP16 transactivation domain.
 23. A gene expression cassette comprising apolynucleotide encoding a hybrid polypeptide comprising atransactivation domain and a retinoid X receptor ligand binding domain,wherein the transactivation domain is from a nuclear receptor other thana retinoid X receptor.
 24. The gene expression cassette according toclaim 22, wherein the transactivation domain is a VP16 transactivationdomain.
 25. A gene expression cassette comprising a polynucleotideencoding a hybrid polypeptide comprising a DNA-binding domain encoded bya polynucleotide comprising a nucleic acid sequence selected from thegroup consisting of a GAL4 DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO:43) and an ecdysone receptor ligand binding domain encoded by apolynucleotide comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,and SEQ ID NO:
 10. 26. A gene expression cassette comprising apolynucleotide encoding a hybrid polypeptide comprising a DNA-bindingdomain comprising an amino acid sequence selected from the groupconsisting of a GAL4 DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44)and an ecdysone receptor ligand binding domain comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:
 20. 27. A geneexpression cassette comprising a polynucleotide encoding a hybridpolypeptide comprising a DNA-binding domain encoded by a polynucleotidecomprising a nucleic acid sequence selected from the group consisting ofa GALA DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO: 43) and a retinoidX receptor ligand binding domain encoded by a polynucleotide comprisinga nucleic acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.28. A gene expression cassette comprising a polynucleotide encoding ahybrid polypeptide comprising a DNA-binding domain comprising an aminoacid sequence selected from the group consisting of a GAL4 DBD (SEQ IDNO: 42) or a LexA DBD (SEQ ID NO: 44) and a retinoid X receptor ligandbinding domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO:
 40. 29. A gene expression cassette comprising apolynucleotide encoding a hybrid polypeptide comprising atransactivation domain encoded by a polynucleotide comprising a nucleicacid sequence of SEQ ID NO: 45 and an ecdysone receptor ligand bindingdomain encoded by a polynucleotide comprising a nucleic acid sequenceselected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10.
 30. A gene expression cassettecomprising a polynucleotide encoding a hybrid polypeptide comprising atransactivation domain comprising an amino acid sequence of SEQ ID NO:46 and an ecdysone receptor ligand binding domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:
 20. 31. Agene expression cassette comprising a polynucleotide encoding a hybridpolypeptide comprising a transactivation domain encoded by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45 and aretinoid X receptor ligand binding domain encoded by a polynucleotidecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQID NO:
 30. 32. A gene expression cassette comprising a polynucleotideencoding a hybrid polypeptide comprising a transactivation domaincomprising an amino acid sequence of SEQ ID NO: 46 and a retinoid Xreceptor ligand binding domain comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO:
 40. 33. An isolatedpolynucleotide encoding an ecdysone receptor polypeptide or a retinoid Xreceptor polypeptide comprising a truncation mutation, wherein thetruncation mutation reduces ligand binding activity of the ecdysonereceptor polypeptide or the retinoid X receptor polypeptide.
 34. Anisolated polynucleotide encoding an ecdysone receptor polypeptide or aretinoid X receptor polypeptide comprising a truncation mutation,wherein the truncation mutation reduces steroid binding activity of theecdysone receptor polypeptide or the retinoid X receptor polypeptide.35. An isolated polynucleotide encoding an ecdysone receptor polypeptideor a retinoid X receptor polypeptide comprising a truncation mutation,wherein the truncation mutation reduces non-steroid binding activity ofthe ecdysone receptor polypeptide or the retinoid X receptorpolypeptide.
 36. An isolated polynucleotide encoding an ecdysonereceptor polypeptide or a retinoid X receptor polypeptide comprising atruncation mutation, wherein the truncation mutation enhances ligandbinding activity of the ecdysone receptor polypeptide or the retinoid Xreceptor polypeptide.
 37. An isolated polynucleotide encoding anecdysone receptor polypeptide or a retinoid X receptor polypeptidecomprising a truncation mutation, wherein the truncation mutationenhances steroid binding activity of the ecdysone receptor polypeptideor the retinoid X receptor polypeptide.
 38. An isolated polynucleotideencoding an ecdysone receptor polypeptide or a retinoid X receptorpolypeptide comprising a truncation mutation, wherein the truncationmutation enhances non-steroid binding activity of the ecdysone receptorpolypeptide or the retinoid X receptor polypeptide.
 39. An isolatedpolynucleotide encoding a retinoid X receptor polypeptide comprising atruncation mutation, wherein the truncation mutation increases ligandsensitivity of the retinoid X receptor polypeptide.
 40. An isolatedpolynucleotide encoding a retinoid X receptor polypeptide comprising atruncation mutation, wherein the truncation mutation increases ligandsensitivity of a heterodimer, wherein the heterodimer comprises saidretinoid X receptor polypeptide and a dimerization partner.
 41. Theisolated polynucleotide according to claim 40, wherein the dimerizationpartner is an ecdysone receptor polypeptide.
 42. An isolatedpolynucleotide encoding a truncated ecdysone receptor polypeptide,wherein the polynucleotide comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, and SEQ ID NO:
 10. 43. An isolated polypeptide encoded bythe isolated polynucleotide according to claim
 42. 44. An isolatedtruncated ecdysone receptor polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:
 20. 45. An isolatedpolynucleotide encoding a truncated retinoid X receptor polypeptide,wherein the polynucleotide comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, and SEQ ID NO:
 30. 46. An isolated polypeptideencoded by the isolated polynucleotide according to claim
 45. 47. Anisolated truncated retinoid X receptor polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO:
 40. 48. Amethod of modulating the expression of a gene in a host cell comprisingthe gene to be modulated comprising the steps of: a) introducing intothe host cell the gene expression modulation system according to claim1; and b) introducing into the host cell a ligand that independentlycombines with the ligand binding domains of the first polypeptide andthe second polypeptide; wherein the gene to be expressed is a componentof a chimeric gene comprising: i) a response element to which the DNAbinding domain from the first polypeptide binds; ii) a promoter that isactivated by the transactivation domain of the second polypeptide; andiii) a gene whose expression is to be modulated, whereby a complex isformed comprising the ligand, the first polypeptide, and the secondpolypeptide, and whereby the complex modulates expression of the gene inthe host cell.
 49. The method according to claim 48, wherein the ligandis a compound of the formula:

wherein: E is a (C₄-C₆)alkyl containing a tertiary carbon or acyano(C₃-C₅)alkyl containing a tertiary carbon; R¹ is H, Me, Et, i-Pr,F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN,C°CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃,CH═CHCN, allyl, azido, SCN, or SCHF₂; R² is H, Me, Et, n-Pr, i-Pr,formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C°CH,1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc,NMe₂, NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃,CH═CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH—CN, orjoined with R³ and the phenyl carbons to which R² and R³ are attached toform an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to aphenyl carbon, or a dihydropyryl ring with the oxygen adjacent to aphenyl carbon; R³ is H, Et, or joined with R² and the phenyl carbons towhich R² and R³ are attached to form an ethylenedioxy, a dihydrofurylring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ringwith the oxygen adjacent to a phenyl carbon; R⁴, R⁵, and R⁶ areindependently H, Me, Et, F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F,CH₂Cl, CH₂OH, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, orSEt.
 50. A method of modulating the expression of a gene in a host cellcomprising the gene to be modulated comprising the steps of: a)introducing into the host cell the gene expression modulation system ofclaim 5; and b) introducing into the host cell a ligand thatindependently combines with the ligand binding domains of the firstpolypeptide and the second polypeptide; wherein the gene to be expressedis a component of a chimeric gene comprising: i) a response element towhich the DNA binding domain from the first polypeptide binds; ii) apromoter that is activated by the transactivation domain of the secondpolypeptide; and iii) a gene whose expression is to be modulated,whereby a complex is formed comprising the ligand, the firstpolypeptide, and the second polypeptide, and whereby the complexmodulates expression of the gene in the host cell.
 51. The methodaccording to claim 50, wherein the ligand is a compound of the formula:

wherein: E is a (C₄-C)alkyl containing a tertiary carbon or acyano(C₃-C₅)alkyl containing a tertiary carbon; R¹ is H, Me, Et, i-Pr,F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN,C°CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃,CH═CHCN, allyl, azido, SCN, or SCHF₂; R² is H, Me, Et, n-Pr, i-Pr,formyl, CF₃, CHF₂, CHCl₇, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C°CH,1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-nPr, OAc,NMe2, NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃,CH═CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH—CN, orjoined with R³ and the phenyl carbons to which R² and R³ are attached toform an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to aphenyl carbon, or a dilydropyryl ring with the oxygen adjacent to aphenyl carbon; R³ is H, Et, or joined with R² and the phenyl carbons towhich R² and R³ are attached to form an ethylenedioxy, a dihydrofurylring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ringwith the oxygen adjacent to a phenyl carbon; R⁴, R⁵, and R⁶ areindependently H, Me, Et, F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F,CH₂Cl, CH₂OH, CN, C°CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, orSEt.
 52. A method of modulating the expression of a gene in a host cellcomprising the gene to be modulated comprising the steps of: a)introducing into the host cell the gene expression modulation system ofclaim 11; and b) introducing into the host cell a ligand thatindependently conibines with the ligand binding domains of the firstpolypeptide and the second polypeptide; wherein the gene to be expressedis a component of a chimeric gene comprising: i) a response element towhich the DNA binding domain from the first polypeptide binds; ii) apromoter that is activated by the transactivation domain of the secondpolypeptide; and iii) a gene whose expression is to be modulated,whereby a complex is formed comprising the ligand, the firstpolypeptide, and the second polypeptide, and whereby the complexmodulates expression of the gene in the host cell.
 53. The methodaccording to claim 52, wherein the ligand is a compound of the formula:

wherein: E is a (C₄-C₆)alkyl containing a tertiary carbon or acyano(C₃-C₅)alkyl containing a tertiary carbon; R¹ is H, Me, Et, i-Pr,F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN,C°CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃,CH═CHCN, allyl, azido, SCN, or SCHF₂; R² is H, Me, Et, n-Pr, i-Pr,formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C°CH,1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-nPr, OAc,NMe₂, NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF2CF3,CH═CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH—CN, orjoined with R³ and the phenyl carbons to which R² and R³ are attached toform an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to aphenyl carbon, or a dihydropyryl ring with the oxygen adjacent to aphenyl carbon; R³ is H, Et, or joined with R² and the phenyl carbons towhich R² and R³ are attached to form an ethylenedioxy, a dihydrofurylring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ringwith the oxygen adjacent to a phenyl carbon; R⁴, R⁵, and R⁶ areindependently H, Me, Et, F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F,CH₂Cl, CH₂OH, CN, C°CH, 1-propynyl, 2-propynyL vinyl, OMe, OEt, SMe, orSEt.
 54. An isolated host cell into which the gene expression modulationsystem according to claim 1 has been introduced.
 55. The isolated hostcell according to claim 54, wherein the host cell is selected from thegroup consisting of abacterial cell, a fungal cell, a yeast cell, aplant cell, an animal cell, and a mammalian cell.
 56. The isolated hostcell according to claim 55, wherein the host cell is a plant cell, amurine cell, or a human cell.
 57. An isolated host cell into which thegene expression modulation system according to claim 5 has beenintroduced.
 58. The isolated host cell according to claim 57, whereinthe host cell is selected from the group consisting of a bacterial cell,a fungal cell, a yeast cell, a plant cell, an animal cell, and amammalian cell.
 59. The isolated host cell according to claim 58,wherein the host cell is a plant cell, a murine cell, or a human cell.60. An isolated host cell into which the gene expression modulationsystem according to claim 11 has been introduced.
 61. The isolated hostcell according to claim 60, wherein the host cell is selected from thegroup consisting of a bacterial cell, a fungal cell, a yeast cell, aplant cell, an animal cell, and a mammalian cell.
 62. The isolated hostcell according to claim 61, wherein the host cell is a plant cell, amurine cell, or a human cell.
 63. A non-human organism comprising a hostcell into which the gene expression modulation system according to claim1 has been introduced.
 64. The non-human organism according to claim 63,wherein the non-human organism is selected from the group consisting ofa bacterium, a fungus, a yeast, a plant, an animal, and a mammal. 65.The non-human organism according to claim 64, wherein the non-humanorganism is selected from the group consisting of a plant, a mouse, arat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep,a monkey, and a chimpanzee.
 66. A non-human organism comprising a hostcell into which the gene expression modulation system according to claimS has been introduced.
 67. The non-human organism according to claim 66,wherein the non-human organism is selected from the group consisting ofa bacterium, a fungus, a yeast, a plant, an animal, and a mammal. 68.The non-human organism according to claim 67, wherein the non-humanorganism is selected from the group consisting of a plant, a mouse, arat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep,a monkey, and a chimpanzee.
 69. A non-human organism comprising a hostcell into which the gene expression modulation system according to claim11 has been introduced.
 70. The non-human organism according to claim69, wherein the non-human organism is selected from the group consistingof a bacterium, a fungus, a yeast, a plant, an animal, and a mammal. 71.The non-human organism according to claim 70, wherein the non-humanorganism is selected from the group consisting of a plant, a mouse, arat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep,a monkey, and a chimpanzee.